Polypeptides Having Cellulolytic Enhancing Activity and Polynucleotides Encoding Same

ABSTRACT

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/439,569, filed Feb. 22, 2017, which is a divisional applicationof U.S. application Ser. No. 15/182,170 filed Jun. 14, 2016, now U.S.Pat. No. 9,605,037, which is a divisional application of U.S.application Ser. No. 14/004,141 filed Mar. 9, 2012, now U.S. Pat. No.9,409,958, which is a 35 U.S.C. §371 national application ofPCT/US2012/028483 filed Mar. 9, 2012, which claims priority or thebenefit under 35 U.S.C. §119 of U.S. Provisional Application Ser. No.61/451,413, filed Mar. 10, 2011, the contents of which are fullyincorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under CooperativeAgreement DE-FC36-08GO18080 awarded by the Department of Energy. Thegovernment has certain rights in this invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to polypeptides having cellulolyticenhancing activity and polynucleotides encoding the polypeptides. Theinvention also relates to nucleic acid constructs, vectors, and hostcells comprising the polynucleotides as well as methods of producing andusing the polypeptides.

Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently linked bybeta-1,4-bonds. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the lignocellulose is converted tofermentable sugars, e.g., glucose, the fermentable sugars are easilyfermented by yeast into ethanol.

WO 2005/074647, WO 2008/148131, and WO 2011/035027 disclose isolatedGH61 polypeptides having cellulolytic enhancing activity and thepolynucleotides thereof from Thielavia terrestris. WO 2005/074656 and WO2010/065830 disclose isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Thermoascusaurantiacus. WO 2007/089290 discloses an isolated GH61 polypeptidehaving cellulolytic enhancing activity and the polynucleotide thereoffrom Trichoderma reesei. WO 2009/085935, WO 2009/085859, WO 2009/085864,and WO 2009/085868 disclose isolated GH61 polypeptides havingcellulolytic enhancing activity and the polynucleotides thereof fromMyceliophthora thermophila. WO 2010/138754 discloses isolated GH61polypeptides having cellulolytic enhancing activity and thepolynucleotides thereof from Aspergillus fumigatus. WO 2011/005867discloses isolated GH61 polypeptides having cellulolytic enhancingactivity and the polynucleotides thereof from Penicillium pinophilum. WO2011/039319 discloses isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Thermoascus sp.WO 2011/041397 discloses isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Penicillium sp.WO 2011/041504 discloses isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Thermoascuscrustaceus. WO 2008/151043 discloses methods of increasing the activityof a GH61 polypeptide having cellulolytic enhancing activity by adding asoluble activating divalent metal cation to a composition comprising thepolypeptide.

The present invention provides GH61 polypeptides having cellulolyticenhancing activity and polynucleotides encoding the polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havingcellulolytic enhancing activity selected from the group consisting of:

(a) a polypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 6 or SEQ ID NO: 8, at least 65% sequenceidentity to the mature polypeptide of SEQ ID NO: 4, at least 70%sequence identity to the mature polypeptide of SEQ ID NO: 10, or atleast 80% sequence identity to the mature polypeptide of SEQ ID NO: 2;

(b) a polypeptide encoded by a polynucleotide that hybridizes undermedium-high, high, or very high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, (ii) the genomic DNA sequence ofthe mature polypeptide coding sequence of SEQ ID NO: 3, SEQ ID NO: 5, orSEQ ID NO: 7, or the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) the full-length complement of (i) or(ii);

(c) a polypeptide encoded by a polynucleotide having at least 60%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 5 or SEQ ID NO: 7, or the genomic DNA sequence thereof; at least 65%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 3 or the genomic DNA sequence thereof; at least 70% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 9; orat least 80% sequence identity to the mature polypeptide coding sequenceof SEQ ID NO: 1 or the cDNA sequence thereof;

(d) a variant of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4,SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10 comprising a substitution,deletion, and/or insertion at one or more (e.g., several) positions; and

(e) a fragment of the polypeptide of (a), (b), (c), or (d) that hascellulolytic enhancing activity.

The present invention also relates to isolated polynucleotides encodingthe polypeptides of the present invention; nucleic acid constructs;recombinant expression vectors; recombinant host cells comprising thepolynucleotides; and methods of producing the polypeptides.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a polypeptide ofthe present invention.

The present invention also relates to methods for producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptide ofthe present invention; (b) fermenting the saccharified cellulosicmaterial with one or more fermenting microorganisms to produce thefermentation product; and (c) recovering the fermentation product fromthe fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore fermenting microorganisms, wherein the cellulosic material issaccharified with an enzyme composition in the presence of a polypeptideof the present invention.

The present invention also relates to a polynucleotide encoding a signalpeptide comprising or consisting of amino acids 1 to 20 of SEQ ID NO: 2,amino acids 1 to 17 of SEQ ID NO: 4, amino acids 1 to 23 of SEQ ID NO:6, amino acids 1 to 16 of SEQ ID NO: 8, or amino acids 1 to 19 of SEQ IDNO: 10, which is operably linked to a gene encoding a protein; nucleicacid constructs, expression vectors, and recombinant host cellscomprising the polynucleotides; and methods of producing a protein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of an Aurantiporus alborubescens Aua2 GH61polypeptide on enzymatic hydrolysis of pretreated corn stover.

FIG. 2 shows the effect of an Aurantiporus alborubescens Aua1 GH61polypeptide on enzymatic hydrolysis of pretreated corn stover.

FIG. 3 shows the effect of a Trichophaea saccata Tsa1 GH61 polypeptideon enzymatic hydrolysis of pretreated corn stover.

FIG. 4 shows the effect of a Trichophaea saccata Tsa2 GH61 polypeptideon enzymatic hydrolysis of pretreated corn stover.

FIG. 5 shows the effect of a Penicillium thomii Pt1 GH61 polypeptide onenzymatic hydrolysis of pretreated corn stover.

DEFINITIONS

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of thepresent invention, acetylxylan esterase activity is determined using 0.5mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20 (polyoxyethylene sorbitan monolaurate). Oneunit of acetylxylan esterase is defined as the amount of enzyme capableof releasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25°C.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of thepresent invention, alpha-L-arabinofuranosidase activity is determinedusing 5 mg of medium viscosity wheat arabinoxylan (MegazymeInternational Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40°C. followed by arabinose analysis by AMINEX® HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. For purposes of the present invention, alpha-glucuronidaseactivity is determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of alpha-glucuronidase equals the amount of enzymecapable of releasing 1 μmole of glucuronic or 4-O-methylglucuronic acidper minute at pH 5, 40° C.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.For purposes of the present invention, beta-glucosidase activity isdetermined using p-nitrophenyl-beta-D-glucopyranoside as substrateaccording to the procedure of Venturi et al., 2002, Extracellularbeta-D-glucosidase from Chaetomium thermophilum var. coprophilum:production, purification and some biochemical properties, J. BasicMicrobiol. 42: 55-66. One unit of beta-glucosidase is defined as 1.0μmole of p-nitrophenolate anion produced per minute at 25° C., pH 4.8from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mMsodium citrate containing 0.01% TWEEN® 20.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides, to remove successive D-xylose residuesfrom non-reducing termini. For purposes of the present invention, oneunit of beta-xylosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20.

Catalytic domain: The term “catalytic domain” means the portion of anenzyme containing the catalytic machinery of the enzyme.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176)that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages incellulose, cellooligosaccharides, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the reducing ornon-reducing ends of the chain (Teeri, 1997, Crystalline cellulosedegradation: New insight into the function of cellobiohydrolases, Trendsin Biotechnology 15: 160-167; Teeri et al., 1998, Trichoderma reeseicellobiohydrolases: why so efficient on crystalline cellulose?, Biochem.Soc. Trans. 26: 173-178). Cellobiohydrolase activity is determinedaccording to the procedures described by Lever et al., 1972, Anal.Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS Letters, 149:152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187: 283-288;and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. In the presentinvention, the Tomme et al. method can be used to determinecellobiohydrolase activity.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic activity include: (1)measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman No1filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-50mg of cellulolytic enzyme protein/g of cellulose in PCS (or otherpretreated cellulosic material) for 3-7 days at a suitable temperature,e.g., 50° C., 55° C., or 60° C., compared to a control hydrolysiswithout addition of cellulolytic enzyme protein. Typical conditions are1 ml reactions, washed or unwashed PCS, 5% insoluble solids, 50 mMsodium acetate pH 5, 1 mM MnSO₄, 50° C., 55° C., or 60° C., 72 hours,sugar analysis by AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA).

Cellulose binding domain: The term “cellulose binding domain” means theportion of an enzyme that mediates binding of the enzyme to amorphousregions of a cellulose substrate. The cellulose binding domain (CBD) isfound either at the N-terminal or at the C-terminal extremity of anenzyme.

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, and wood (including forestryresidue) (see, for example, Wiselogel et al., 1995, in Handbook onBioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis,Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd,1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier etal., 1999, Recent Progress in Bioconversion of Lignocellulosics, inAdvances in Biochemical Engineering/Biotechnology, T. Scheper, managingeditor, Volume 65, pp. 23-40, Springer-Verlag, New York). It isunderstood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is any biomass material. In another preferredaspect, the cellulosic material is lignocellulose, which comprisescellulose, hemicelluloses, and lignin.

In one aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is herbaceous material(including energy crops). In another aspect, the cellulosic material ismunicipal solid waste. In another aspect, the cellulosic material ispulp and paper mill residue. In another aspect, the cellulosic materialis waste paper. In another aspect, the cellulosic material is wood(including forestry residue).

In another aspect, the cellulosic material is arundo. In another aspect,the cellulosic material is bagasse. In another aspect, the cellulosicmaterial is bamboo. In another aspect, the cellulosic material is corncob. In another aspect, the cellulosic material is corn fiber. Inanother aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is orange peel. In another aspect, the cellulosicmaterial is rice straw. In another aspect, the cellulosic material isswitchgrass. In another aspect, the cellulosic material is wheat straw.

In another aspect, the cellulosic material is aspen. In another aspect,the cellulosic material is eucalyptus. In another aspect, the cellulosicmaterial is fir. In another aspect, the cellulosic material is pine. Inanother aspect, the cellulosic material is poplar. In another aspect,the cellulosic material is spruce. In another aspect, the cellulosicmaterial is willow.

In another aspect, the cellulosic material is algal cellulose. Inanother aspect, the cellulosic material is bacterial cellulose. Inanother aspect, the cellulosic material is cotton linter. In anotheraspect, the cellulosic material is filter paper. In another aspect, thecellulosic material is microcrystalline cellulose. In another aspect,the cellulosic material is phosphoric-acid treated cellulose.

In another aspect, the cellulosic material is an aquatic biomass. Asused herein the term “aquatic biomass” means biomass produced in anaquatic environment by a photosynthesis process. The aquatic biomass canbe algae, emergent plants, floating-leaf plants, or submerged plants.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Endoglucanase: The term “endoglucanase” means anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such ascereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696. The enzymes in this family wereoriginally classified as a glycoside hydrolase family based onmeasurement of very weak endo-1,4-beta-D-glucanase activity in onefamily member. The structure and mode of action of these enzymes arenon-canonical and they cannot be considered as bona fide glycosidases.However, they are kept in the CAZy classification on the basis of theircapacity to enhance the breakdown of lignocellulose when used inconjunction with a cellulase or a mixture of cellulases.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)groups from esterified sugar, which is usually arabinose in naturalbiomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate).Feruloyl esterase is also known as ferulic acid esterase,hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA,cinnAE, FAE-I, or FAE-II. For purposes of the present invention,feruloyl esterase activity is determined using 0.5 mMp-nitrophenylferulate as substrate in 50 mM sodium acetate pH 5.0. Oneunit of feruloyl esterase equals the amount of enzyme capable ofreleasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25° C.

Fragment: The term “fragment” means a polypeptide or a domain thereofhaving one or more (e.g., several) amino acids absent from the aminoand/or carboxyl terminus of a mature polypeptide or a domain thereof;wherein the fragment has cellulolytic enhancing activity or cellulosebinding activity. In one aspect, a fragment contains at least 255 aminoacid residues, e.g., at least 270 amino acid residues or at least 285amino acid residues of the mature polypeptide of SEQ ID NO: 2. Inanother aspect, a fragment contains at least at least 185 amino acidresidues, e.g., at least 195 amino acid residues or at least 205 aminoacid residues of the mature polypeptide of SEQ ID NO: 4. In anotheraspect, a fragment contains at least 180 amino acid residues, e.g., atleast 190 amino acid residues or at least 200 amino acid residues of themature polypeptide of SEQ ID NO: 6. In another aspect, a fragmentcontains at least 190 amino acid residues, e.g., at least 200 amino acidresidues or at least 210 amino acid residues of the mature polypeptideof SEQ ID NO: 8. In another aspect, a fragment contains at least 385amino acid residues, e.g., at least 410 amino acid residues or at least435 amino acid residues of the mature polypeptide of SEQ ID NO: 10.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom, D.and Shoham, Y. Microbial hemicellulases. Current Opinion InMicrobiology, 2003, 6(3): 219-228). Hemicellulases are key components inthe degradation of plant biomass. Examples of hemicellulases include,but are not limited to, an acetylmannan esterase, an acetylxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase. The substrates of these enzymes, the hemicelluloses, are aheterogeneous group of branched and linear polysaccharides that arebound via hydrogen bonds to the cellulose microfibrils in the plant cellwall, crosslinking them into a robust network. Hemicelluloses are alsocovalently attached to lignin, forming together with cellulose a highlycomplex structure. The variable structure and organization ofhemicelluloses require the concerted action of many enzymes for itscomplete degradation. The catalytic modules of hemicellulases are eitherglycoside hydrolases (GHs) that hydrolyze glycosidic bonds, orcarbohydrate esterases (CEs), which hydrolyze ester linkages of acetateor ferulic acid side groups. These catalytic modules, based on homologyof their primary sequence, can be assigned into GH and CE families. Somefamilies, with an overall similar fold, can be further grouped intoclans, marked alphabetically (e.g., GH-A). A most informative andupdated classification of these and other carbohydrate active enzymes isavailable in the Carbohydrate-Active Enzymes (CAZy) database.Hemicellulolytic enzyme activities can be measured according to Ghoseand Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752, at a suitabletemperature, e.g., 50° C., 55° C., or 60° C., and pH, e.g., 5.0 or 5.5.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., recombinantproduction in a host cell; multiple copies of a gene encoding thesubstance; and use of a stronger promoter than the promoter naturallyassociated with the gene encoding the substance).

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 21 to 322 of SEQ ID NO: 2 based on theSignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6) thatpredicts amino acids 1 to 20 of SEQ ID NO: 2 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 18 to 234 of SEQID NO: 4 based on the SignalP program that predicts amino acids 1 to 17of SEQ ID NO: 4 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 24 to 233 of SEQ ID NO: 6 based on theSignalP program that predicts amino acids 1 to 23 of SEQ ID NO: 6 are asignal peptide. In another aspect, the mature polypeptide is amino acids17 to 237 of SEQ ID NO: 8 based on the SignalP program that predictsamino acids 1 to 16 of SEQ ID NO: 8 are a signal peptide. In anotheraspect, the mature polypeptide is amino acids 20 to 484 of SEQ ID NO: 10based on the SignalP program that predicts amino acids 1 to 19 of SEQ IDNO: 10 are a signal peptide. It is known in the art that a host cell mayproduce a mixture of two of more different mature polypeptides (i.e.,with a different C-terminal and/or N-terminal amino acid) expressed bythe same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving cellulolytic enhancing activity. In one aspect, the maturepolypeptide coding sequence is nucleotides 61 to 966 of SEQ ID NO: 1 orthe cDNA sequence thereof based on the SignalP program (Nielsen et al.,1997, supra) that predicts nucleotides 1 to 60 of SEQ ID NO: 1 encode asignal peptide. In another aspect, the mature polypeptide codingsequence is nucleotides 52 to 702 of SEQ ID NO: 3 or the genomic DNAsequence thereof based on the SignalP program that predicts nucleotides1 to 51 of SEQ ID NO: 3 encode a signal peptide. In another aspect, themature polypeptide coding sequence is nucleotides 70 to 699 of SEQ IDNO: 5 or the genomic DNA sequence thereof based on the SignalP programthat predicts nucleotides 1 to 69 of SEQ ID NO: 5 encode a signalpeptide. In another aspect, the mature polypeptide coding sequence isnucleotides 49 to 711 of SEQ ID NO: 7 or the genomic DNA sequencethereof based on the SignalP program that predicts nucleotides 1 to 48of SEQ ID NO: 7 encode a signal peptide. In another aspect, the maturepolypeptide coding sequence is nucleotides 58 to 1452 of SEQ ID NO: 9based on the SignalP program that predicts nucleotides 1 to 57 of SEQ IDNO: 9 encode a signal peptide.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 60° C.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Polypeptide having cellulolytic enhancing activity: The term“polypeptide having cellulolytic enhancing activity” means a GH61polypeptide that catalyzes the enhancement of the hydrolysis of acellulosic material by enzyme having cellulolytic activity. For purposesof the present invention, cellulolytic enhancing activity is determinedby measuring the increase in reducing sugars or the increase of thetotal of cellobiose and glucose from the hydrolysis of a cellulosicmaterial by cellulolytic enzyme under the following conditions: 1-50 mgof total protein/g of cellulose in PCS, wherein total protein iscomprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/wprotein of a GH61 polypeptide having cellulolytic enhancing activity for1-7 days at a suitable temperature, e.g., 50° C., 55° C., or 60° C., andpH, e.g., 5.0 or 5.5, compared to a control hydrolysis with equal totalprotein loading without cellulolytic enhancing activity (1-50 mg ofcellulolytic protein/g of cellulose in PCS). In a preferred aspect, amixture of CELLUCLAST® 1.5L (Novozymes A/S, Bagsvaerd, Denmark) in thepresence of 2-3% of total protein weight Aspergillus oryzaebeta-glucosidase (recombinantly produced in Aspergillus oryzae accordingto WO 02/095014) or 2-3% of total protein weight Aspergillus fumigatusbeta-glucosidase (recombinantly produced in Aspergillus oryzae asdescribed in WO 2002/095014) of cellulase protein loading is used as thesource of the cellulolytic activity.

The GH61 polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold, at least1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 10-fold, or at least 20-fold.

The polypeptides of the present invention have at least 20%, e.g., atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, and at least 100% of the cellulolytic enhancingactivity of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.

Pretreated corn stover: The term “PCS” or “Pretreated Corn Stover” meansa cellulosic material derived from corn stover by treatment with heatand dilute sulfuric acid, alkaline pretreatment, or neutralpretreatment.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the -nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having cellulolytic enhancing activity. In one aspect, asubsequence contains at least 765 nucleotides, e.g., at leastnucleotides 810 or at least nucleotides 855 of SEQ ID NO: 1; or the cDNAsequence thereof. In another aspect, a subsequence contains at least 555nucleotides, e.g., at least nucleotides 585 or at least nucleotides 615of SEQ ID NO: 3; or the genomic DNA sequence thereof. In another aspect,a subsequence contains at least 540 nucleotides, e.g., at leastnucleotides 570 or at least nucleotides 600 of SEQ ID NO: 5; or thegenomic DNA sequence thereof. In another aspect, a subsequence containsat least 570 nucleotides, e.g., at least nucleotides 600 or at leastnucleotides 630 of SEQ ID NO: 7; or the genomic DNA sequence thereof. Inanother aspect, a subsequence contains at least 1155 nucleotides, e.g.,at least nucleotides 1230 or at least nucleotides 1305 of SEQ ID NO: 9.

Variant: The term “variant” means a polypeptide having cellulolyticenhancing activity comprising an alteration, i.e., a substitution,insertion, and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, Recent progress in the assays of xylanolytic enzymes, 2006,Journal of the Science of Food and Agriculture 86(11): 1636-1647;Spanikova and Biely, 2006, Glucuronoyl esterase—Novel carbohydrateesterase produced by Schizophyllum commune, FEBS Letters 580(19):4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek,1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctionalbeta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. The most common total xylanolytic activity assay is basedon production of reducing sugars from polymeric 4-O-methylglucuronoxylan as described in Bailey, Biely, Poutanen, 1992,Interlaboratory testing of methods for assay of xylanase activity,Journal of Biotechnology 23(3): 257-270. Xylanase activity can also bedetermined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON®X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200mM sodium phosphate buffer pH 6 at 37° C. One unit of xylanase activityis defined as 1.0 μmole of azurine produced per minute at 37° C., pH 6from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6buffer.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, A new reaction for colorimetric determination ofcarbohydrates, Anal. Biochem 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. For purposes of the present invention, xylanaseactivity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01%TRITON® X-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unitof xylanase activity is defined as 1.0 μmole of azurine produced perminute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate pH 6 buffer.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides Having CellulolyticEnhancing Activity

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 6 orSEQ ID NO: 8 of at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100%; the mature polypeptide of SEQID NO: 4 of at least 65%, e.g., at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%; the mature polypeptide of SEQ ID NO: 10 ofat least 70%, e.g., at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%; orthe mature polypeptide of SEQ ID NO: 2 of at least 80%, e.g., at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%; which have cellulolytic enhancing activity. In one aspect,the polypeptides differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, or SEQ ID NO: 10 or an allelic variant thereof; or is afragment thereof having cellulolytic enhancing activity. In anotheraspect, the polypeptide comprises or consists of the mature polypeptideof SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO:10. In another preferred aspect, the polypeptide comprises or consistsof amino acids 21 to 322 of SEQ ID NO: 2, amino acids 18 to 234 of SEQID NO: 4, amino acids 24 to 233 of SEQ ID NO: 6, amino acids 17 to 237of SEQ ID NO: 8, or amino acids 20 to 484 of SEQ ID NO: 10.

In another embodiment, the present invention relates to isolatedpolypeptides having cellulolytic enhancing activity that are encoded bypolynucleotides that hybridize under very low stringency conditions, lowstringency conditions, medium stringency conditions, medium-highstringency conditions, high stringency conditions, or very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9,(ii) the genomic DNA sequence of the mature polypeptide coding sequenceof SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, or the cDNA sequence ofthe mature polypeptide coding sequence of SEQ ID NO: 1, or (iii) thefull-length complement of (i) or (ii) (Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.)

The polynucleotide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, or SEQ ID NO: 9, or a subsequence thereof, as well as thepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,or SEQ ID NO: 10, or a fragment thereof, may be used to design nucleicacid probes to identify and clone DNA encoding polypeptides havingcellulolytic enhancing activity from strains of different genera orspecies according to methods well known in the art. In particular, suchprobes can be used for hybridization with the genomic DNA or cDNA of acell of interest, following standard Southern blotting procedures, inorder to identify and isolate the corresponding gene therein. Suchprobes can be considerably shorter than the entire sequence, but shouldbe at least 15, e.g., at least 25, at least 35, or at least 70nucleotides in length. Preferably, the nucleic acid probe is at least100 nucleotides in length, e.g., at least 200 nucleotides, at least 300nucleotides, at least 400 nucleotides, at least 500 nucleotides, atleast 600 nucleotides, at least 700 nucleotides, at least 800nucleotides, or at least 900 nucleotides in length. Both DNA and RNAprobes can be used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having cellulolytic enhancing activity. Genomic orother DNA from such other strains may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that hybridizes with SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9 or a subsequencethereof, the carrier material is preferably used in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO:9; the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; the genomic DNA sequence ofthe mature polypeptide coding sequence of SEQ ID NO: 3, SEQ ID NO: 5, orSEQ ID NO: 7, or the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 1; or a full-length complement thereof; or asubsequence thereof; under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions can be detected using, for example, X-ray film or any otherdetection means known in the art.

In one aspect, the nucleic acid probe is a polynucleotide that encodesthe polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, or SEQ ID NO: 10; the mature polypeptide thereof; or a fragmentthereof. In another aspect, the nucleic acid probe is SEQ ID NO: 1 orthe cDNA sequence thereof, SEQ ID NO: 9, or SEQ ID NO: 3, SEQ ID NO: 5,or SEQ ID NO: 7, or the genomic DNA sequence thereof.

In another embodiment, the present invention relates to isolatedpolypeptides having cellulolytic enhancing activity encoded bypolynucleotides having a sequence identity to the mature polypeptidecoding sequence of SEQ ID NO: 5 or SEQ ID NO: 7, or the genomic DNAsequence thereof, of at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%; the mature polypeptidecoding sequence of SEQ ID NO: 3 or the genomic DNA sequence thereof, ofat least 65%, e.g., at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%; the mature polypeptide coding sequence of SEQ ID NO: 9 ofat least 70%, e.g., at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%; orthe mature polypeptide coding sequence of SEQ ID NO: 1 or the cDNAsequence thereof of at least 80%, e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, or SEQ ID NO: 10 comprising a substitution, deletion, and/orinsertion at one or more (e.g., several) positions. In an embodiment,the number of amino acid substitutions, deletions and/or insertionsintroduced into the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4,SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10 is up to 10, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, or 10. The amino acid changes may be of a minornature, that is conservative amino acid substitutions or insertions thatdo not significantly affect the folding and/or activity of the protein;small deletions, typically of 1-30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to 20-25 residues; or a smallextension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for cellulolytic enhancing activity to identifyamino acid residues that are critical to the activity of the molecule.See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The activesite of the enzyme or other biological interaction can also bedetermined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity ofessential amino acids can also be inferred from an alignment with arelated polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Sources of Polypeptides Having Cellulolytic Enhancing Activity

A polypeptide having cellulolytic enhancing activity of the presentinvention may be obtained from microorganisms of any genus. For purposesof the present invention, the term “obtained from” as used herein inconnection with a given source shall mean that the polypeptide encodedby a polynucleotide is produced by the source or by a strain in whichthe polynucleotide from the source has been inserted. In one aspect, thepolypeptide obtained from a given source is secreted extracellularly.

The polypeptide may be a fungal polypeptide. For example, thepolypeptide may be a yeast polypeptide such as a Candida, Kluyveromyces,Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; ora filamentous fungal polypeptide such as an Acremonium, Agaricus,Alternaria, Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis,Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis,Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia,Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex,Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide.

In another aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis polypeptide.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium inops,Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporiummerdarium, Chrysosporium pannicola, Chrysosporium queenslandicum,Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa,Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptide.

In another aspect, the polypeptide is an Aurantiporus alborubescenspolypeptide, e.g., a polypeptide obtained from Aurantiporusalborubescens NN008024.

In another aspect, the polypeptide is an Trichophaea saccatapolypeptide, e.g., a polypeptide obtained from Trichophaea saccata CBS804.70.

In another aspect, the polypeptide is a Penicillium thomii polypeptide,e.g., a polypeptide obtained from Penicillium thomii IBT 10776.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Polynucleotides

The present invention also relates to isolated polynucleotides encodingpolypeptides of the present invention, as described herein.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNAor cDNA, or a combination thereof. The cloning of the polynucleotidesfrom genomic DNA can be effected, e.g., by using the well knownpolymerase chain reaction (PCR) or antibody screening of expressionlibraries to detect cloned DNA fragments with shared structuralfeatures. See, e.g., Innis et al., 1990, PCR: A Guide to Methods andApplication, Academic Press, New York. Other nucleic acid amplificationprocedures such as ligase chain reaction (LCR), ligation activatedtranscription (LAT) and polynucleotide-based amplification (NASBA) maybe used. The polynucleotides may be cloned from a strain ofAurantiporus, Trichophaea, or Penicillium, or a related organism andthus, for example, may be an allelic or species variant of thepolypeptide encoding region of the polynucleotide.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for synthesizing polypeptides substantiallysimilar to the polypeptide. The term “substantially similar” to thepolypeptide refers to non-naturally occurring forms of the polypeptide.These polypeptides may differ in some engineered way from thepolypeptide isolated from its native source, e.g., variants that differin specific activity, thermostability, pH optimum, or the like. Thevariants may be constructed on the basis of the polynucleotide presentedas the mature polypeptide coding sequence of SEQ ID NO: 1 or the cDNAsequence thereof, SEQ ID NO: 9, or SEQ ID NO: 3, SEQ ID NO: 5, or SEQ IDNO: 7, or the genomic DNA thereof, e.g., a subsequence thereof, and/orby introduction of nucleotide substitutions that do not result in achange in the amino acid sequence of the polypeptide, but whichcorrespond to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionsthat may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of the polypeptide. Manipulation of the polynucleotideprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifyingpolynucleotides utilizing recombinant DNA methods are well known in theart.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and mutant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus nigerglucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysequences in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter,and Trichoderma reesei cellobiohydrolase II promoter may be used. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding thepolypeptide would be operably linked to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, adeA(phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB(phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris a hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausi, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and optionally (b) recovering thepolypeptide. In one aspect, the cell is an Aurantiporus cell. In anotheraspect, the cell is an Aurantiporus alborubescens cell. In anotheraspect, the cell is Aurantiporus alborubescens strain NN00802. Inanother aspect, the cell is a Trichophaea cell. In another aspect, thecell is a Trichophaea saccata cell. In another aspect, the cell isTrichophaea saccata CBS 804.70. In another aspect, the cell is aPenicillium cell. In another aspect, the cell is a Penicillium thomiicell. In another aspect, the cell is Penicillium thomii IBT 10776.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and optionally (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cells may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. In one aspect, a fermentation broth comprising thepolypeptide is recovered.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising a polynucleotideof the present invention so as to express and produce a polypeptide ordomain in recoverable quantities. The polypeptide or domain may berecovered from the plant or plant part. Alternatively, the plant orplant part containing the polypeptide or domain may be used as such forimproving the quality of a food or feed, e.g., improving nutritionalvalue, palatability, and rheological properties, or to destroy anantinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing the polypeptide or domainmay be constructed in accordance with methods known in the art. Inshort, the plant or plant cell is constructed by incorporating one ormore expression constructs encoding the polypeptide or domain into theplant host genome or chloroplast genome and propagating the resultingmodified plant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide or domain operablylinked with appropriate regulatory sequences required for expression ofthe polynucleotide in the plant or plant part of choice. Furthermore,the expression construct may comprise a selectable marker useful foridentifying plant cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide or domainis desired to be expressed. For instance, the expression of the geneencoding a polypeptide or domain may be constitutive or inducible, ormay be developmental, stage or tissue specific, and the gene product maybe targeted to a specific tissue or plant part such as seeds or leaves.Regulatory sequences are, for example, described by Tague et al., 1988,Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or therice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be induced by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide or domain in the plant. For instance, thepromoter enhancer element may be an intron that is placed between thepromoter and the polynucleotide encoding a polypeptide or domain. Forinstance, Xu et al., 1993, supra, disclose the use of the first intronof the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transformingmonocots, although other transformation methods may be used for theseplants. A method for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al., 1993, Plant Mol. Biol.21: 415-428. Additional transformation methods include those describedin U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are hereinincorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct of the present invention, transgenic plants may be made bycrossing a plant having the construct to a second plant lacking theconstruct. For example, a construct encoding a polypeptide or domain canbe introduced into a particular plant variety by crossing, without theneed for ever directly transforming a plant of that given variety.Therefore, the present invention encompasses not only a plant directlyregenerated from cells which have been transformed in accordance withthe present invention, but also the progeny of such plants. As usedherein, progeny may refer to the offspring of any generation of a parentplant prepared in accordance with the present invention. Such progenymay include a DNA construct prepared in accordance with the presentinvention. Crossing results in the introduction of a transgene into aplant line by cross pollinating a starting line with a donor plant line.Non-limiting examples of such steps are described in U.S. Pat. No.7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a polypeptideof the present invention comprising: (a) cultivating a transgenic plantor a plant cell comprising a polynucleotide encoding the polypeptideunder conditions conducive for production of the polypeptide or domain;and (b) recovering the polypeptide.

Removal or Reduction of Cellulolytic Enhancing Activity

The present invention also relates to methods of producing a mutant of aparent cell, which comprises disrupting or deleting a polynucleotide, ora portion thereof, encoding a polypeptide of the present invention,which results in the mutant cell producing less of the polypeptide thanthe parent cell when cultivated under the same conditions.

The mutant cell may be constructed by reducing or eliminating expressionof the polynucleotide using methods well known in the art, for example,insertions, disruptions, replacements, or deletions. In a preferredaspect, the polynucleotide is inactivated. The polynucleotide to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required forexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thepolynucleotide. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the polynucleotide may be performed bysubjecting the parent cell to mutagenesis and selecting for mutant cellsin which expression of the polynucleotide has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the polynucleotide may be accomplishedby insertion, substitution, or deletion of one or more nucleotides inthe gene or a regulatory element required for transcription ortranslation thereof. For example, nucleotides may be inserted or removedso as to result in the introduction of a stop codon, the removal of thestart codon, or a change in the open reading frame. Such modification orinactivation may be accomplished by site-directed mutagenesis or PCRgenerated mutagenesis in accordance with methods known in the art.Although, in principle, the modification may be performed in vivo, i.e.,directly on the cell expressing the polynucleotide to be modified, it ispreferred that the modification be performed in vitro as exemplifiedbelow.

An example of a convenient way to eliminate or reduce expression of apolynucleotide is based on techniques of gene replacement, genedeletion, or gene disruption. For example, in the gene disruptionmethod, a nucleic acid sequence corresponding to the endogenouspolynucleotide is mutagenized in vitro to produce a defective nucleicacid sequence that is then transformed into the parent cell to produce adefective gene. By homologous recombination, the defective nucleic acidsequence replaces the endogenous polynucleotide. It may be desirablethat the defective polynucleotide also encodes a marker that may be usedfor selection of transformants in which the polynucleotide has beenmodified or destroyed. In an aspect, the polynucleotide is disruptedwith a selectable marker such as those described herein.

The present invention also relates to methods of inhibiting theexpression of a polypeptide having cellulolytic enhancing activity in acell, comprising administering to the cell or expressing in the cell adouble-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises asubsequence of a polynucleotide of the present invention. In a preferredaspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length.

The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA(miRNA). In a preferred aspect, the dsRNA is small interfering RNA forinhibiting transcription. In another preferred aspect, the dsRNA ismicro RNA for inhibiting translation.

The present invention also relates to such double-stranded RNA (dsRNA)molecules, comprising a portion of the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, orSEQ ID NO: 9 for inhibiting expression of the polypeptide in a cell.While the present invention is not limited by any particular mechanismof action, the dsRNA can enter a cell and cause the degradation of asingle-stranded RNA (ssRNA) of similar or identical sequences, includingendogenous mRNAs. When a cell is exposed to dsRNA, mRNA from thehomologous gene is selectively degraded by a process called RNAinterference (RNAi).

The dsRNAs of the present invention can be used in gene-silencing. Inone aspect, the invention provides methods to selectively degrade RNAusing a dsRNAi of the present invention. The process may be practiced invitro, ex vivo, or in vivo. In one aspect, the dsRNA molecules can beused to generate a loss-of-function mutation in a cell, an organ or ananimal. Methods for making and using dsRNA molecules to selectivelydegrade RNA are well known in the art; see, for example, U.S. Pat. Nos.6,489,127; 6,506,559; 6,511,824; and 6,515,109.

The present invention further relates to a mutant cell of a parent cellthat comprises a disruption or deletion of a polynucleotide encoding thepolypeptide or a control sequence thereof or a silenced gene encodingthe polypeptide, which results in the mutant cell producing less of thepolypeptide or no polypeptide compared to the parent cell.

The polypeptide-deficient mutant cells are particularly useful as hostcells for expression of native and heterologous polypeptides. Therefore,the present invention further relates to methods of producing a nativeor heterologous polypeptide, comprising: (a) cultivating the mutant cellunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide. The term “heterologous polypeptides” meanspolypeptides that are not native to the host cell, e.g., a variant of anative protein. The host cell may comprise more than one copy of apolynucleotide encoding the native or heterologous polypeptide.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallycellulolytic enhancing-free product are of particular interest in theproduction of eukaryotic polypeptides, in particular fungal proteinssuch as enzymes. The cellulolytic enhancing-deficient cells may also beused to express heterologous proteins of pharmaceutical interest such ashormones, growth factors, receptors, and the like. The term “eukaryoticpolypeptides” includes not only native polypeptides, but also thosepolypeptides, e.g., enzymes, which have been modified by amino acidsubstitutions, deletions or additions, or other such modifications toenhance activity, thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from cellulolytic enhancing activity that is producedby a method of the present invention.

Fermentation Broth Formulations or Cell Compositions

The present invention also relates to a fermentation broth formulationor a cell composition comprising a polypeptide of the present invention.The fermentation broth product further comprises additional ingredientsused in the fermentation process, such as, for example, cells(including, the host cells containing the gene encoding the polypeptideof the present invention which are used to produce the polypeptide ofinterest), cell debris, biomass, fermentation media and/or fermentationproducts. In some embodiments, the composition is a cell-killed wholebroth containing organic acid(s), killed cells and/or cell debris, andculture medium.

The term “fermentation broth” as used herein refers to a preparationproduced by cellular fermentation that undergoes no or minimal recoveryand/or purification. For example, fermentation broths are produced whenmicrobial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes by host cells) and secretion into cell culture medium. Thefermentation broth can contain unfractionated or fractionated contentsof the fermentation materials derived at the end of the fermentation.Typically, the fermentation broth is unfractionated and comprises thespent culture medium and cell debris present after the microbial cells(e.g., filamentous fungal cells) are removed, e.g., by centrifugation.In some embodiments, the fermentation broth contains spent cell culturemedium, extracellular enzymes, and viable and/or nonviable microbialcells.

In an embodiment, the fermentation broth formulation and cellcompositions comprise a first organic acid component comprising at leastone 1-5 carbon organic acid and/or a salt thereof and a second organicacid component comprising at least one 6 or more carbon organic acidand/or a salt thereof. In a specific embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or a mixture of two or more of the foregoing and the second organic acidcomponent is benzoic acid, cyclohexanecarboxylic acid, 4-methylvalericacid, phenylacetic acid, a salt thereof, or a mixture of two or more ofthe foregoing.

In one aspect, the composition contains an organic acid(s), andoptionally further contains killed cells and/or cell debris. In oneembodiment, the killed cells and/or cell debris are removed from acell-killed whole broth to provide a composition that is free of thesecomponents.

The fermentation broth formulations or cell compostions may furthercomprise a preservative and/or anti-microbial (e.g., bacteriostatic)agent, including, but not limited to, sorbitol, sodium chloride,potassium sorbate, and others known in the art.

The cell-killed whole broth or composition may contain theunfractionated contents of the fermentation materials derived at the endof the fermentation. Typically, the cell-killed whole broth orcomposition contains the spent culture medium and cell debris presentafter the microbial cells (e.g., filamentous fungal cells) are grown tosaturation, incubated under carbon-limiting conditions to allow proteinsynthesis (e.g., expression of cellulase and/or glucosidase enzyme(s)).In some embodiments, the cell-killed whole broth or composition containsthe spent cell culture medium, extracellular enzymes, and killedfilamentous fungal cells. In some embodiments, the microbial cellspresent in the cell-killed whole broth or composition can bepermeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically aliquid, but may contain insoluble components, such as killed cells, celldebris, culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedliquid composition.

The whole broth formulations and cell compositions of the presentinvention may be produced by a method described in WO 90/15861 or WO2010/096673.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Enzyme Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that thecellulolytic enhancing activity of the composition has been increased,e.g., with an enrichment factor of at least 1.1.

The compositions may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the compositions may comprise multiple enzymaticactivities, such as one or more (e.g., several) enzymes selected fromthe group consisting of a cellulase, a polypeptide having cellulolyticenhancing activity, a hemicellulase, an esterase, an expansin, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Thecompositions may be stabilized in accordance with methods known in theart.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Uses

The present invention is also directed to the following methods forusing the polypeptides having cellulolytic enhancing activity, orcompositions thereof.

The present invention also relates to methods for degrading a cellulosicmaterial, comprising: treating the cellulosic material with an enzymecomposition in the presence of a polypeptide having cellulolyticenhancing activity of the present invention. In one aspect, the methodsfurther comprise recovering the degraded or converted cellulosicmaterial. Soluble products of degradation or conversion of thecellulosic material can be separated from insoluble cellulosic materialusing a method known in the art such as, for example, centrifugation,filtration, or gravity settling.

The present invention also relates to methods of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity of the present invention; (b)fermenting the saccharified cellulosic material with one or more (e.g.,several) fermenting microorganisms to produce the fermentation product;and (c) recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (e.g., several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition in the presence of apolypeptide having cellulolytic enhancing activity of the presentinvention. In one aspect, the fermenting of the cellulosic materialproduces a fermentation product. In another aspect, the methods furthercomprise recovering the fermentation product from the fermentation.

The methods of the present invention can be used to saccharify thecellulosic material to fermentable sugars and to convert the fermentablesugars to many useful fermentation products, e.g., fuel, potableethanol, and/or platform chemicals (e.g., acids, alcohols, ketones,gases, and the like). The production of a desired fermentation productfrom the cellulosic material typically involves pretreatment, enzymatichydrolysis (saccharification), and fermentation.

The processing of the cellulosic material according to the presentinvention can be accomplished using methods conventional in the art.Moreover, the methods of the present invention can be implemented usingany conventional biomass processing apparatus configured to operate inaccordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and co-fermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF);and direct microbial conversion (DMC), also sometimes calledconsolidated bioprocessing (CBP). SHF uses separate process steps tofirst enzymatically hydrolyze the cellulosic material to fermentablesugars, e.g., glucose, cellobiose, and pentose monomers, and thenferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of the cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the co-fermentation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog. 15: 817-827).HHF involves a separate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more (e.g.,several) steps where the same organism is used to produce the enzymesfor conversion of the cellulosic material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd, L. R.,Weimer, P. J., van Zyl, W. H., and Pretorius, I. S., 2002, Microbialcellulose utilization: Fundamentals and biotechnology, Microbiol. Mol.Biol. Reviews 66: 506-577). It is understood herein that any methodknown in the art comprising pretreatment, enzymatic hydrolysis(saccharification), fermentation, or a combination thereof, can be usedin the practicing the methods of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Fernandade Castilhos Corazza, Flávio Faria de Moraes, Gisella Maria Zanin andIvo Neitzel, 2003, Optimal control in fed-batch reactor for thecellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov,A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysisof cellulose: 1. A mathematical model for a batch reactor process, Enz.Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee,J. M., 1983, Bioconversion of waste cellulose by using an attritionbioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensivestirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn,A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996,Enhancement of enzymatic cellulose hydrolysis using a novel type ofbioreactor with intensive stirring induced by electromagnetic field,Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor typesinclude fluidized bed, upflow blanket, immobilized, and extruder typereactors for hydrolysis and/or fermentation.

Pretreatment. In practicing the methods of the present invention, anypretreatment process known in the art can be used to disrupt plant cellwall components of the cellulosic material (Chandra et al., 2007,Substrate pretreatment: The key to effective enzymatic hydrolysis oflignocellulosics?, Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbeand Zacchi, 2007, Pretreatment of lignocellulosic materials forefficient bioethanol production, Adv. Biochem. Engin./Biotechnol. 108:41-65; Hendriks and Zeeman, 2009, Pretreatments to enhance thedigestibility of lignocellulosic biomass, Bioresource Technol. 100:10-18; Mosier et al., 2005, Features of promising technologies forpretreatment of lignocellulosic biomass, Bioresource Technol. 96:673-686; Taherzadeh and Karimi, 2008, Pretreatment of lignocellulosicwastes to improve ethanol and biogas production: A review, Int. J. ofMol. Sci. 9: 1621-1651; Yang and Wyman, 2008, Pretreatment: the key tounlocking low-cost cellulosic ethanol, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, sieving, pre-soaking, wetting, washing, and/or conditioningprior to pretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, ionic liquid, and gammairradiation pretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment. In steam pretreatment, the cellulosic material isheated to disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. The cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably performed at 140-250° C., e.g., 160-200° C. or 170-190° C.,where the optimal temperature range depends on addition of a chemicalcatalyst. Residence time for the steam pretreatment is preferably 1-60minutes, e.g., 1-30 minutes, 1-20 minutes, 3-12 minutes, or 4-10minutes, where the optimal residence time depends on temperature rangeand addition of a chemical catalyst. Steam pretreatment allows forrelatively high solids loadings, so that the cellulosic material isgenerally only moist during the pretreatment. The steam pretreatment isoften combined with an explosive discharge of the material after thepretreatment, which is known as steam explosion, that is, rapid flashingto atmospheric pressure and turbulent flow of the material to increasethe accessible surface area by fragmentation (Duff and Murray, 1996,Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl.Microbiol. Biotechnol. 59: 618-628; U.S. Patent Application No.20020164730). During steam pretreatment, hemicellulose acetyl groups arecleaved and the resulting acid autocatalyzes partial hydrolysis of thehemicellulose to monosaccharides and oligosaccharides. Lignin is removedto only a limited extent.

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Such a pretreatment can convertcrystalline cellulose to amorphous cellulose. Examples of suitablechemical pretreatment processes include, for example, dilute acidpretreatment, lime pretreatment, wet oxidation, ammonia fiber/freezeexplosion (AFEX), ammonia percolation (APR), ionic liquid, andorganosolv pretreatments.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 5% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (Ballesteroset al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006,Enzyme Microb. Technol. 39: 756-762). In dilute acid pretreatment, thecellulosic material is mixed with dilute acid, typically H₂SO₄, andwater to form a slurry, heated by steam to the desired temperature, andafter a residence time flashed to atmospheric pressure. The dilute acidpretreatment can be performed with a number of reactor designs, e.g.,plug-flow reactors, counter-current reactors, or continuouscounter-current shrinking bed reactors (Duff and Murray, 1996, supra;Schell et al., 2004, Bioresource Technol. 91: 179-188; Lee et al., 1999,Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to,sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), andammonia fiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium oxide or calcium hydroxideat temperatures of 85-150° C. and residence times from 1 hour to severaldays (Wyman et al., 2005, Bioresource Technol. 96: 1959-1966; Mosier etal., 2005, Bioresource Technol. 96: 673-686). WO 2006/110891, WO2006/110899, WO 2006/110900, and WO 2006/110901 disclose pretreatmentmethods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed preferably at 1-40% drymatter, e.g., 2-30% dry matter or 5-20% dry matter, and often theinitial pH is increased by the addition of alkali such as sodiumcarbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion) can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves treating the cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-150°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96:2014-2018). During AFEX pretreatment cellulose and hemicelluloses remainrelatively intact. Lignin-carbohydrate complexes are cleaved.

Organosolv pretreatment delignifies the cellulosic material byextraction using aqueous ethanol (40-60% ethanol) at 160-200° C. for30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan etal., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl.Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose andlignin is removed.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as adilute acid treatment, and more preferably as a continuous dilute acidtreatment. The acid is typically sulfuric acid, but other acids can alsobe used, such as acetic acid, citric acid, nitric acid, phosphoric acid,tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof.Mild acid treatment is conducted in the pH range of preferably 1-5,e.g., 1-4 or 1-2.5. In one aspect, the acid concentration is in therange from preferably 0.01 to 10 wt % acid, e.g., 0.05 to 5 wt % acid or0.1 to 2 wt % acid. The acid is contacted with the cellulosic materialand held at a temperature in the range of preferably 140-200° C., e.g.,165-190° C., for periods ranging from 1 to 60 minutes.

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the cellulosic material is present duringpretreatment in amounts preferably between 10-80 wt %, e.g., 20-70 wt %or 30-60 wt %, such as around 40 wt %. The pretreated cellulosicmaterial can be unwashed or washed using any method known in the art,e.g., washed with water.

Mechanical Pretreatment or Physical Pretreatment: The term “mechanicalpretreatment” or “physical pretreatment” refers to any pretreatment thatpromotes size reduction of particles. For example, such pretreatment caninvolve various types of grinding or milling (e.g., dry milling, wetmilling, or vibratory ball milling).

The cellulosic material can be pretreated both physically (mechanically)and chemically. Mechanical or physical pretreatment can be coupled withsteaming/steam explosion, hydrothermolysis, dilute or mild acidtreatment, high temperature, high pressure treatment, irradiation (e.g.,microwave irradiation), or combinations thereof. In one aspect, highpressure means pressure in the range of preferably about 100 to about400 psi, e.g., about 150 to about 250 psi. In another aspect, hightemperature means temperatures in the range of about 100 to about 300°C., e.g., about 140 to about 200° C. In a preferred aspect, mechanicalor physical pretreatment is performed in a batch-process using a steamgun hydrolyzer system that uses high pressure and high temperature asdefined above, e.g., a Sunds Hydrolyzer available from Sunds DefibratorAB, Sweden. The physical and chemical pretreatments can be carried outsequentially or simultaneously, as desired.

Accordingly, in a preferred aspect, the cellulosic material is subjectedto physical (mechanical) or chemical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms and/or enzymes (see, for example,Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212; Ghosh and Singh, 1993, Physicochemical andbiological treatments for enzymatic/microbial conversion of cellulosicbiomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994,Pretreating lignocellulosic biomass: a review, in Enzymatic Conversionof Biomass for Fuels Production, Himmel, M. E., Baker, J. O., andOverend, R. P., eds., ACS Symposium Series 566, American ChemicalSociety, Washington, D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson andHahn-Hagerdal, 1996, Fermentation of lignocellulosic hydrolysates forethanol production, Enz. Microb. Tech. 18: 312-331; and Vallander andEriksson, 1990, Production of ethanol from lignocellulosic materials:State of the art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification. In the hydrolysis step, also known assaccharification, the cellulosic material, e.g., pretreated, ishydrolyzed to break down cellulose and/or hemicellulose to fermentablesugars, such as glucose, cellobiose, xylose, xylulose, arabinose,mannose, galactose, and/or soluble oligosaccharides. The hydrolysis isperformed enzymatically by an enzyme composition in the presence of apolypeptide having cellulolytic enhancing activity of the presentinvention. The enzymes of the compositions can be added simultaneouslyor sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In one aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme(s), i.e., optimal forthe enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the cellulosic material is fed gradually to,for example, an enzyme containing hydrolysis solution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 120 hours, e.g., about 16 to about 72 hours or about24 to about 48 hours. The temperature is in the range of preferablyabout 25° C. to about 70° C., e.g., about 30° C. to about 65° C., about40° C. to about 60° C., or about 50° C. to about 55° C. The pH is in therange of preferably about 3 to about 8, e.g., about 3.5 to about 7,about 4 to about 6, or about 5.0 to about 5.5. The dry solids content isin the range of preferably about 5 to about 50 wt %, e.g., about 10 toabout 40 wt % or about 20 to about 30 wt %.

The enzyme compositions can comprise any protein useful in degrading thecellulosic material.

In one aspect, the enzyme composition comprises or further comprises oneor more (e.g., several) proteins selected from the group consisting of acellulase, a GH61 polypeptide having cellulolytic enhancing activity, ahemicellulase, an esterase, an expansin, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin. Inanother aspect, the cellulase is preferably one or more (e.g., several)enzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase. In another aspect, thehemicellulase is preferably one or more (e.g., several) enzymes selectedfrom the group consisting of an acetylmannan esterase, an acetylxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase.

In another aspect, the enzyme composition comprises one or more (e.g.,several) cellulolytic enzymes. In another aspect, the enzyme compositioncomprises or further comprises one or more (e.g., several)hemicellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (e.g., several) cellulolytic enzymes and one ormore (e.g., several) hemicellulolytic enzymes. In another aspect, theenzyme composition comprises one or more (e.g., several) enzymesselected from the group of cellulolytic enzymes and hemicellulolyticenzymes. In another aspect, the enzyme composition comprises anendoglucanase. In another aspect, the enzyme composition comprises acellobiohydrolase. In another aspect, the enzyme composition comprises abeta-glucosidase. In another aspect, the enzyme composition comprises apolypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises an endoglucanase and a polypeptidehaving cellulolytic enhancing activity. In another aspect, the enzymecomposition comprises a cellobiohydrolase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises a beta-glucosidase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises an endoglucanase and a cellobiohydrolase. Inanother aspect, the enzyme composition comprises an endoglucanase and abeta-glucosidase. In another aspect, the enzyme composition comprises acellobiohydrolase and a beta-glucosidase. In another aspect, the enzymecomposition comprises an endoglucanase, a cellobiohydrolase, and apolypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises an endoglucanase, a beta-glucosidase,and a polypeptide having cellulolytic enhancing activity. In anotheraspect, the enzyme composition comprises a cellobiohydrolase, abeta-glucosidase, and a polypeptide having cellulolytic enhancingactivity. In another aspect, the enzyme composition comprises anendoglucanase, a cellobiohydrolase, and a beta-glucosidase. In anotheraspect, the enzyme composition comprises an endoglucanase, acellobiohydrolase, a beta-glucosidase, and a polypeptide havingcellulolytic enhancing activity.

In another aspect, the enzyme composition comprises an acetylmannanesterase. In another aspect, the enzyme composition comprises anacetylxylan esterase. In another aspect, the enzyme compositioncomprises an arabinanase (e.g., alpha-L-arabinanase). In another aspect,the enzyme composition comprises an arabinofuranosidase (e.g.,alpha-L-arabinofuranosidase). In another aspect, the enzyme compositioncomprises a coumaric acid esterase. In another aspect, the enzymecomposition comprises a feruloyl esterase. In another aspect, the enzymecomposition comprises a galactosidase (e.g., alpha-galactosidase and/orbeta-galactosidase). In another aspect, the enzyme composition comprisesa glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, theenzyme composition comprises a glucuronoyl esterase. In another aspect,the enzyme composition comprises a mannanase. In another aspect, theenzyme composition comprises a mannosidase (e.g., beta-mannosidase). Inanother aspect, the enzyme composition comprises a xylanase. In apreferred aspect, the xylanase is a Family 10 xylanase. In anotheraspect, the enzyme composition comprises a xylosidase (e.g.,beta-xylosidase).

In another aspect, the enzyme composition comprises an esterase. Inanother aspect, the enzyme composition comprises an expansin. In anotheraspect, the enzyme composition comprises a laccase. In another aspect,the enzyme composition comprises a ligninolytic enzyme. In a preferredaspect, the ligninolytic enzyme is a manganese peroxidase. In anotherpreferred aspect, the ligninolytic enzyme is a lignin peroxidase. Inanother preferred aspect, the ligninolytic enzyme is a H₂O₂-producingenzyme. In another aspect, the enzyme composition comprises a pectinase.In another aspect, the enzyme composition comprises a peroxidase. Inanother aspect, the enzyme composition comprises a protease. In anotheraspect, the enzyme composition comprises a swollenin.

In the methods of the present invention, the enzyme(s) can be addedprior to or during saccharification, saccharification and fermentation,or fermentation.

One or more (e.g., several) components of the enzyme composition may bewild-type proteins, recombinant proteins, or a combination of wild-typeproteins and recombinant proteins. For example, one or more (e.g.,several) components may be native proteins of a cell, which is used as ahost cell to express recombinantly one or more (e.g., several) othercomponents of the enzyme composition. One or more (e.g., several)components of the enzyme composition may be produced as monocomponents,which are then combined to form the enzyme composition. The enzymecomposition may be a combination of multicomponent and monocomponentprotein preparations.

The enzymes used in the methods of the present invention may be in anyform suitable for use, such as, for example, a fermentation brothformulation or a cell composition, a cell lysate with or withoutcellular debris, a semi-purified or purified enzyme preparation, or ahost cell as a source of the enzymes. The enzyme composition may be adry powder or granulate, a non-dusting granulate, a liquid, a stabilizedliquid, or a stabilized protected enzyme. Liquid enzyme preparationsmay, for instance, be stabilized by adding stabilizers such as a sugar,a sugar alcohol or another polyol, and/or lactic acid or another organicacid according to established methods.

The optimum amounts of the enzymes and polypeptides having cellulolyticenhancing activity depend on several factors including, but not limitedto, the mixture of component cellulolytic enzymes and/orhemicellulolytic enzymes, the cellulosic material, the concentration ofcellulosic material, the pretreatment(s) of the cellulosic material,temperature, time, pH, and inclusion of fermenting organism (e.g., yeastfor Simultaneous Saccharification and Fermentation).

In one aspect, an effective amount of cellulolytic or hemicellulolyticenzyme to the cellulosic material is about 0.5 to about 50 mg, e.g.,about 0.5 to about 40 mg, about 0.5 to about 25 mg, about 0.75 to about20 mg, about 0.75 to about 15 mg, about 0.5 to about 10 mg, or about 2.5to about 10 mg per g of the cellulosic material.

In another aspect, an effective amount of a polypeptide havingcellulolytic enhancing activity to the cellulosic material is about 0.01to about 50.0 mg, e.g., about 0.01 to about 40 mg, about 0.01 to about30 mg, about 0.01 to about 20 mg, about 0.01 to about 10 mg, about 0.01to about 5 mg, about 0.025 to about 1.5 mg, about 0.05 to about 1.25 mg,about 0.075 to about 1.25 mg, about 0.1 to about 1.25 mg, about 0.15 toabout 1.25 mg, or about 0.25 to about 1.0 mg per g of the cellulosicmaterial.

In another aspect, an effective amount of a polypeptide havingcellulolytic enhancing activity to cellulolytic or hemicellulolyticenzyme is about 0.005 to about 1.0 g, e.g., about 0.01 to about 1.0 g,about 0.15 to about 0.75 g, about 0.15 to about 0.5 g, about 0.1 toabout 0.5 g, about 0.1 to about 0.25 g, or about 0.05 to about 0.2 g perg of cellulolytic or hemicellulolytic enzyme.

The polypeptides having cellulolytic enzyme activity or hemicellulolyticenzyme activity as well as other proteins/polypeptides useful in thedegradation of the cellulosic material, e.g., GH61 polypeptides havingcellulolytic enhancing activity (collectively hereinafter “polypeptideshaving enzyme activity”) can be derived or obtained from any suitableorigin, including, bacterial, fungal, yeast, plant, or mammalian origin.The term “obtained” also means herein that the enzyme may have beenproduced recombinantly in a host organism employing methods describedherein, wherein the recombinantly produced enzyme is either native orforeign to the host organism or has a modified amino acid sequence,e.g., having one or more (e.g., several) amino acids that are deleted,inserted and/or substituted, i.e., a recombinantly produced enzyme thatis a mutant and/or a fragment of a native amino acid sequence or anenzyme produced by nucleic acid shuffling processes known in the art.Encompassed within the meaning of a native enzyme are natural variantsand within the meaning of a foreign enzyme are variants obtainedrecombinantly, such as by site-directed mutagenesis or shuffling.

A polypeptide having enzyme activity may be a bacterial polypeptide. Forexample, the polypeptide may be a Gram positive bacterial polypeptidesuch as a Bacillus, Streptococcus, Streptomyces, Staphylococcus,Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus,Caldicellulosiruptor, Acidothermus, Thermobifidia, or Oceanobacilluspolypeptide having enzyme activity, or a Gram negative bacterialpolypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter,Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, orUreaplasma polypeptide having enzyme activity.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans polypeptide having enzyme activity.

The polypeptide having enzyme activity may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide having enzyme activity; or more preferably a filamentousfungal polypeptide such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having enzymeactivity.

In one aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis polypeptide having enzyme activity.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus,Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum,Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporiummerdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielaviaspededonium, Thielavia setosa, Thielavia subthermophila, Thielaviaterrestris, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichophaeasaccata polypeptide having enzyme activity.

Chemically modified or protein engineered mutants of polypeptides havingenzyme activity may also be used.

One or more (e.g., several) components of the enzyme composition may bea recombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host is preferably a heterologous host (enzyme isforeign to host), but the host may under certain conditions also be ahomologous host (enzyme is native to host). Monocomponent cellulolyticproteins may also be prepared by purifying such a protein from afermentation broth.

In one aspect, the one or more (e.g., several) cellulolytic enzymescomprise a commercial cellulolytic enzyme preparation. Examples ofcommercial cellulolytic enzyme preparations suitable for use in thepresent invention include, for example, CELLIC® CTec (Novozymes A/S),CELLIC® CTec2 (Novozymes A/S), CELLIC® CTec3 (Novozymes A/S),CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188 (Novozymes A/S), CELLUZYME™(Novozymes A/S), CEREFLO™ (Novozymes A/S), and ULTRAFLO™ (NovozymesA/S), ACCELERASE™ (Genencor Int.), LAMINEX™ (Genencor Int.), SPEZYME™ CP(Genencor Int.), FILTRASE® NL (DSM); METHAPLUS® S/L 100 (DSM), ROHAMENT™7069 W (Röhm GmbH), FIBREZYME® LDI (Dyadic International, Inc.),FIBREZYME® LBR (Dyadic International, Inc.), or VISCOSTAR® 150L (DyadicInternational, Inc.). The cellulase enzymes are added in amountseffective from about 0.001 to about 5.0 wt % of solids, e.g., about0.025 to about 4.0 wt % of solids or about 0.005 to about 2.0 wt % ofsolids.

Examples of bacterial endoglucanases that can be used in the methods ofthe present invention, include, but are not limited to, an Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention, include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263, Trichodermareesei Cel7B endoglucanase I (GENBANK™ accession no. M15665),Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene63:11-22), Trichoderma reesei Cel5A endoglucanase II (GENBANK™ accessionno. M19373), Trichoderma reesei endoglucanase Ill (Okada et al., 1988,Appl. Environ. Microbiol. 64: 555-563, GENBANK™ accession no. AB003694),Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, MolecularMicrobiology 13: 219-228, GENBANK™ accession no. Z33381), Aspergillusaculeatus endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18:5884), Aspergillus kawachii endoglucanase (Sakamoto et al., 1995,Current Genetics 27: 435-439), Erwinia carotovara endoglucanase(Saarilahti et al., 1990, Gene 90: 9-14), Fusarium oxysporumendoglucanase (GENBANK™ accession no. L29381), Humicola grisea var.thermoidea endoglucanase (GENBANK™ accession no. AB003107), Melanocarpusalbomyces endoglucanase (GENBANK™ accession no. MAL515703), Neurosporacrassa endoglucanase (GENBANK™ accession no. XM_324477), Humicolainsolens endoglucanase V, Myceliophthora thermophila CBS 117.65endoglucanase, basidiomycete CBS 495.95 endoglucanase, basidiomycete CBS494.95 endoglucanase, Thielavia terrestris NRRL 8126 CEL6Bendoglucanase, Thielavia terrestris NRRL 8126 CEL6C endoglucanase,Thielavia terrestris NRRL 8126 CEL7C endoglucanase, Thielavia terrestrisNRRL 8126 CEL7E endoglucanase, Thielavia terrestris NRRL 8126 CEL7Fendoglucanase, Cladorrhinum foecundissimum ATCC 62373 CEL7Aendoglucanase, and Trichoderma reesei strain No. VTT-D-80133endoglucanase (GENBANK™ accession no. M15665).

Examples of cellobiohydrolases useful in the present invention include,but are not limited to, Aspergillus aculeatus cellobiohydrolase II (WO2011/059740), Chaetomium thermophilum cellobiohydrolase I, Chaetomiumthermophilum cellobiohydrolase II, Humicola insolens cellobiohydrolaseI, Myceliophthora thermophila cellobiohydrolase II (WO 2009/042871),Thielavia hyrcanie cellobiohydrolase II (WO 2010/141325), Thielaviaterrestris cellobiohydrolase II (CEL6A, WO 2006/074435), Trichodermareesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, andTrichophaea saccata cellobiohydrolase II (WO 2010/057086).

Examples of beta-glucosidases useful in the present invention include,but are not limited to, beta-glucosidases from Aspergillus aculeatus(Kawaguchi et al., 1996, Gene 173: 287-288), Aspergillus fumigatus (WO2005/047499), Aspergillus niger (Dan et al., 2000, J. Biol. Chem. 275:4973-4980), Aspergillus oryzae (WO 2002/095014), Penicillium brasilianumIBT 20888 (WO 2007/019442 and WO 2010/088387), Thielavia terrestris (WO2011/035029), and Trichophaea saccata (WO 2007/019442).

The beta-glucosidase may be a fusion protein. In one aspect, thebeta-glucosidase is an Aspergillus oryzae beta-glucosidase variant BGfusion protein (WO 2008/057637) or an Aspergillus oryzaebeta-glucosidase fusion protein (WO 2008/057637).

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidasesare disclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat B., 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

Other cellulolytic enzymes that may be used in the present invention aredescribed in WO 98/13465, WO 98/015619, WO 98/015633, WO 99/06574, WO99/10481, WO 99/025847, WO 99/031255, WO 2002/101078, WO 2003/027306, WO2003/052054, WO 2003/052055, WO 2003/052056, WO 2003/052057, WO2003/052118, WO 2004/016760, WO 2004/043980, WO 2004/048592, WO2005/001065, WO 2005/028636, WO 2005/093050, WO 2005/093073, WO2006/074005, WO 2006/117432, WO 2007/071818, WO 2007/071820, WO2008/008070, WO 2008/008793, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,648,263, and U.S. Pat. No. 5,686,593.

In the methods of the present invention, any GH61 polypeptide havingcellulolytic enhancing activity can be used as a component of the enzymecomposition.

Examples of GH61 polypeptides having cellulolytic enhancing activityuseful in the methods of the present invention include, but are notlimited to, GH61 polypeptides from Thielavia terrestris (WO 2005/074647,WO 2008/148131, and WO 2011/035027), Thermoascus aurantiacus (WO2005/074656 and WO 2010/065830), Trichoderma reesei (WO 2007/089290),Myceliophthora thermophila (WO 2009/085935, WO 2009/085859, WO2009/085864, WO 2009/085868), Aspergillus fumigatus (WO 2010/138754),GH61 polypeptides from Penicillium pinophilum (WO 2011/005867),Thermoascus sp. (WO 2011/039319), Penicillium sp. (WO 2011/041397), andThermoascus crustaceous (WO 2011/041504).

In one aspect, the GH61 polypeptide having cellulolytic enhancingactivity is used in the presence of a soluble activating divalent metalcation according to WO 2008/151043, e.g., manganese sulfate.

In another aspect, the GH61 polypeptide having cellulolytic enhancingactivity is used in the presence of a dioxy compound, a bicyliccompound, a heterocyclic compound, a nitrogen-containing compound, aquinone compound, a sulfur-containing compound, or a liquor obtainedfrom a pretreated cellulosic material such as pretreated corn stover(PCS).

The dioxy compound may include any suitable compound containing two ormore oxygen atoms. In some aspects, the dioxy compounds contain asubstituted aryl moiety as described herein. The dioxy compounds maycomprise one or more (e.g., several) hydroxyl and/or hydroxylderivatives, but also include substituted aryl moieties lacking hydroxyland hydroxyl derivatives. Non-limiting examples of the dioxy compoundsinclude pyrocatechol or catechol; caffeic acid; 3,4-dihydroxybenzoicacid; 4-tert-butyl-5-methoxy-1,2-benzenediol; pyrogallol; gallic acid;methyl-3,4,5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone;2,6-dimethoxyphenol; sinapinic acid; 3,5-dihydroxybenzoic acid;4-chloro-1,2-benzenediol; 4-nitro-1,2-benzenediol; tannic acid; ethylgallate; methyl glycolate; dihydroxyfumaric acid; 2-butyne-1,4-diol;(croconic acid; 1,3-propanediol; tartaric acid; 2,4-pentanediol;3-ethyoxy-1,2-propanediol; 2,4,4′-trihydroxybenzophenone;cis-2-butene-1,4-diol; 3,4-dihydroxy-3-cyclobutene-1,2-dione;dihydroxyacetone; acrolein acetal; methyl-4-hydroxybenzoate;4-hydroxybenzoic acid; and methyl-3,5-dimethoxy-4-hydroxybenzoate; or asalt or solvate thereof.

The bicyclic compound may include any suitable substituted fused ringsystem as described herein. The compounds may comprise one or more(e.g., several) additional rings, and are not limited to a specificnumber of rings unless otherwise stated. In one aspect, the bicycliccompound is a flavonoid. In another aspect, the bicyclic compound is anoptionally substituted isoflavonoid. In another aspect, the bicycliccompound is an optionally substituted flavylium ion, such as anoptionally substituted anthocyanidin or optionally substitutedanthocyanin, or derivative thereof. Non-limiting examples of thebicyclic compounds include epicatechin; quercetin; myricetin; taxifolin;kaempferol; morin; acacetin; naringenin; isorhamnetin; apigenin;cyanidin; cyanin; kuromanin; keracyanin; or a salt or solvate thereof.

The heterocyclic compound may be any suitable compound, such as anoptionally substituted aromatic or non-aromatic ring comprising aheteroatom, as described herein. In one aspect, the heterocyclic is acompound comprising an optionally substituted heterocycloalkyl moiety oran optionally substituted heteroaryl moiety. In another aspect, theoptionally substituted heterocycloalkyl moiety or optionally substitutedheteroaryl moiety is an optionally substituted 5-memberedheterocycloalkyl or an optionally substituted 5-membered heteroarylmoiety. In another aspect, the optionally substituted heterocycloalkylor optionally substituted heteroaryl moiety is an optionally substitutedmoiety selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl,oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl,thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl, thianaphthenyl,carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl,quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl,benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisazolyl,dimethylhydantoin, pyrazinyl, tetrahydrofuranyl, pyrrolinyl,pyrrolidinyl, morpholinyl, indolyl, diazepinyl, azepinyl, thiepinyl,piperidinyl, and oxepinyl. In another aspect, the optionally substitutedheterocycloalkyl moiety or optionally substituted heteroaryl moiety isan optionally substituted furanyl. Non-limiting examples of theheterocyclic compounds include(1,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one;4-hydroxy-5-methyl-3-furanone; 5-hydroxy-2(5H)-furanone;[1,2-dihydroxyethyl]furan-2,3,4(5H)-trione; α-hydroxy-γ-butyrolactone;ribonic γ-lactone; aldohexuronicaldohexuronic acid γ-lactone; gluconicacid δ-lactone; 4-hydroxycoumarin; dihydrobenzofuran;5-(hydroxymethyl)furfural; furoin; 2(5H)-furanone;5,6-dihydro-2H-pyran-2-one; and5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one; or a salt or solvatethereof.

The nitrogen-containing compound may be any suitable compound with oneor more nitrogen atoms. In one aspect, the nitrogen-containing compoundcomprises an amine, imine, hydroxylamine, or nitroxide moiety.Non-limiting examples of the nitrogen-containing compounds includeacetone oxime; violuric acid; pyridine-2-aldoxime; 2-aminophenol;1,2-benzenediamine; 2,2,6,6-tetramethyl-1-piperidinyloxy;5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterine; andmaleamic acid; or a salt or solvate thereof.

The quinone compound may be any suitable compound comprising a quinonemoiety as described herein. Non-limiting examples of the quinonecompounds include 1,4-benzoquinone; 1,4-naphthoquinone;2-hydroxy-1,4-naphthoquinone; 2,3-dimethoxy-5-methyl-1,4-benzoquinone orcoenzyme Q₀; 2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone;1,4-dihydroxyanthraquinone; 3-hydroxy-1-methyl-5,6-indolinedione oradrenochrome; 4-tert-butyl-5-methoxy-1,2-benzoquinone; pyrroloquinolinequinone; or a salt or solvate thereof.

The sulfur-containing compound may be any suitable compound comprisingone or more sulfur atoms. In one aspect, the sulfur-containing comprisesa moiety selected from thionyl, thioether, sulfinyl, sulfonyl,sulfamide, sulfonamide, sulfonic acid, and sulfonic ester. Non-limitingexamples of the sulfur-containing compounds include ethanethiol;2-propanethiol; 2-propene-1-thiol; 2-mercaptoethanesulfonic acid;benzenethiol; benzene-1,2-dithiol; cysteine; methionine; glutathione;cystine; or a salt or solvate thereof.

In one aspect, an effective amount of such a compound described above tocellulosic material as a molar ratio to glucosyl units of cellulose isabout 10⁻⁶ to about 10, e.g., about 10⁻⁶ to about 7.5, about 10⁻⁶ toabout 5, about 10⁻⁶ to about 2.5, about 10⁻⁶ to about 1, about 10⁻⁶ toabout 1, about 10⁻⁶ to about 10⁻¹, about 10⁻⁴ to about 10⁻¹, about 10⁻³to about 10⁻¹, or about 10⁻³ to about 10⁻². In another aspect, aneffective amount of such a compound described above is about 0.1 μM toabout 1 M, e.g., about 0.5 μM to about 0.75 M, about 0.75 μM to about0.5 M, about 1 μM to about 0.25 M, about 1 μM to about 0.1 M, about 5 μMto about 50 mM, about 10 μM to about 25 mM, about 50 μM to about 25 mM,about 10 μM to about 10 mM, about 5 μM to about 5 mM, or about 0.1 mM toabout 1 mM.

The term “liquor” means the solution phase, either aqueous, organic, ora combination thereof, arising from treatment of a lignocellulose and/orhemicellulose material in a slurry, or monosaccharides thereof, e.g.,xylose, arabinose, mannose, etc., under conditions as described herein,and the soluble contents thereof. A liquor for cellulolytic enhancementof a GH61 polypeptide can be produced by treating a lignocellulose orhemicellulose material (or feedstock) by applying heat and/or pressure,optionally in the presence of a catalyst, e.g., acid, optionally in thepresence of an organic solvent, and optionally in combination withphysical disruption of the material, and then separating the solutionfrom the residual solids. Such conditions determine the degree ofcellulolytic enhancement obtainable through the combination of liquorand a GH61 polypeptide during hydrolysis of a cellulosic substrate by acellulase preparation. The liquor can be separated from the treatedmaterial using a method standard in the art, such as filtration,sedimentation, or centrifugation.

In one aspect, an effective amount of the liquor to cellulose is about10⁻⁶ to about 10 g per g of cellulose, e.g., about 10⁻⁶ to about 7.5 g,about 10⁻⁶ to about 5 g, about 10⁻⁶ to about 2.5 g, about 10⁻⁶ to about1 g, about 10⁻⁶ to about 1 g, about 10⁻⁶ to about 10⁻¹ g, about 10⁻⁴ toabout 10⁻¹ g, about 10⁻³ to about 10⁻¹ g, or about 10⁻³ to about 10⁻² gper g of cellulose.

In one aspect, the one or more (e.g., several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes A/S),CELLIC® HTec (Novozymes A/S), CELLIC® HTec2 (Novozymes A/S), CELLIC®HTec3 (Novozymes A/S), VISCOZYME® (Novozymes A/S), ULTRAFLO® (NovozymesA/S), PULPZYME® HC (Novozymes A/S), MULTIFECT® Xylanase (Genencor),ACCELLERASE® XY (Genencor), ACCELLERASE® XC (Genencor), ECOPULP® TX-200A(AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit,Wales, UK), DEPOL™ 740L. (Biocatalysts Limit, Wales, UK), and DEPOL™762P (Biocatalysts Limit, Wales, UK).

Examples of xylanases useful in the methods of the present inventioninclude, but are not limited to, xylanases from Aspergillus aculeatus(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus (WO2006/078256), Penicillium pinophilum (WO 2011/041405), Penicillium sp.(WO 2010/126772), Thielavia terrestris NRRL 8126 (WO 2009/079210), andTrichophaea saccata GH10 (WO 2011/057083).

Examples of beta-xylosidases useful in the methods of the presentinvention include, but are not limited to, beta-xylosidases fromNeurospora crassa (SwissProt accession number Q7SOW4), Trichodermareesei (UniProtKB/TrEMBL accession number Q92458), and Talaromycesemersonii (SwissProt accession number Q8X212).

Examples of acetylxylan esterases useful in the methods of the presentinvention include, but are not limited to, acetylxylan esterases fromAspergillus aculeatus (WO 2010/108918), Chaetomium globosum (Uniprotaccession number Q2GWX4), Chaetomium gracile (GeneSeqP accession numberAAB82124), Humicola insolens DSM 1800 (WO 2009/073709), Hypocreajecorina (WO 2005/001036), Myceliophtera thermophila (WO 2010/014880),Neurospora crassa (UniProt accession number q7s259), Phaeosphaerianodorum (Uniprot accession number Q0UHJ1), and Thielavia terrestris NRRL8126 (WO 2009/042846).

Examples of feruloyl esterases (ferulic acid esterases) useful in themethods of the present invention include, but are not limited to,feruloyl esterases form Humicola insolens DSM 1800 (WO 2009/076122),Neosartorya fischeri (UniProt Accession number A1D9T4), Neurosporacrassa (UniProt accession number Q9HGR3), Penicillium aurantiogriseum(WO 2009/127729), and Thielavia terrestris (WO 2010/053838 and WO2010/065448).

Examples of arabinofuranosidases useful in the methods of the presentinvention include, but are not limited to, arabinofuranosidases fromAspergillus niger (GeneSeqP accession number AAR94170), Humicolainsolens DSM 1800 (WO 2006/114094 and WO 2009/073383), and M. giganteus(WO 2006/114094).

Examples of alpha-glucuronidases useful in the methods of the presentinvention include, but are not limited to, alpha-glucuronidases fromAspergillus clavatus (UniProt accession number alcc12), Aspergillusfumigatus (SwissProt accession number Q4WW45), Aspergillus niger(Uniprot accession number Q96WX9), Aspergillus terreus (SwissProtaccession number Q0CJP9), Humicola insolens (WO 2010/014706),Penicillium aurantiogriseum (WO 2009/068565), Talaromyces emersonii(UniProt accession number Q8X211), and Trichoderma reesei (Uniprotaccession number Q99024).

The polypeptides having enzyme activity used in the methods of thepresent invention may be produced by fermentation of the above-notedmicrobial strains on a nutrient medium containing suitable carbon andnitrogen sources and inorganic salts, using procedures known in the art(see, e.g., Bennett, J. W. and LaSure, L. (eds.), More GeneManipulations in Fungi, Academic Press, C A, 1991). Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). Temperature ranges and other conditions suitable for growthand enzyme production are known in the art (see, e.g., Bailey, J. E.,and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill BookCompany, N Y, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme or protein. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe enzyme to be expressed or isolated. The resulting enzymes producedby the methods described above may be recovered from the fermentationmedium and purified by conventional procedures.

Fermentation. The fermentable sugars obtained from the hydrolyzedcellulosic material can be fermented by one or more (e.g., several)fermenting microorganisms capable of fermenting the sugars directly orindirectly into a desired fermentation product. “Fermentation” or“fermentation process” refers to any fermentation process or any processcomprising a fermentation step. Fermentation processes also includefermentation processes used in the consumable alcohol industry (e.g.,beer and wine), dairy industry (e.g., fermented dairy products), leatherindustry, and tobacco industry. The fermentation conditions depend onthe desired fermentation product and fermenting organism and can easilybe determined by one skilled in the art.

In the fermentation step, sugars, released from the cellulosic materialas a result of the pretreatment and enzymatic hydrolysis steps, arefermented to a product, e.g., ethanol, by a fermenting organism, such asyeast. Hydrolysis (saccharification) and fermentation can be separate orsimultaneous, as described herein.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be hexose and/or pentose fermenting organisms, or acombination thereof. Both hexose and pentose fermenting organisms arewell known in the art. Suitable fermenting microorganisms are able toferment, i.e., convert, sugars, such as glucose, xylose, xylulose,arabinose, maltose, mannose, galactose, and/or oligosaccharides,directly or indirectly into the desired fermentation product. Examplesof bacterial and fungal fermenting organisms producing ethanol aredescribed by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.

Examples of fermenting microorganisms that can ferment hexose sugarsinclude bacterial and fungal organisms, such as yeast. Preferred yeastincludes strains of Candida, Kluyveromyces, and Saccharomyces, e.g.,Candida sonorensis, Kluyveromyces marxianus, and Saccharomycescerevisiae.

Examples of fermenting organisms that can ferment pentose sugars intheir native state include bacterial and fungal organisms, such as someyeast. Preferred xylose fermenting yeast include strains of Candida,preferably C. sheatae or C. sonorensis; and strains of Pichia,preferably P. stipitis, such as P. stipitis CBS 5773. Preferred pentosefermenting yeast include strains of Pachysolen, preferably P.tannophilus. Organisms not capable of fermenting pentose sugars, such asxylose and arabinose, may be genetically modified to do so by methodsknown in the art.

Examples of bacteria that can efficiently ferment hexose and pentose toethanol include, for example, Bacillus coagulans, Clostridiumacetobutylicum, Clostridium thermocellum, Clostridium phytofermentans,Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonasmobilis (Philippidis, 1996, supra).

Other fermenting organisms include strains of Bacillus, such as Bacilluscoagulans; Candida, such as C. sonorensis, C. methanosorbosa, C.diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C.entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis,and C. scehatae; Clostridium, such as C. acetobutylicum, C.thermocellum, and C. phytofermentans; E. coli, especially E. colistrains that have been genetically modified to improve the yield ofethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala;Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K.lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such asS. pombe; Thermoanaerobacter, such as Thermoanaerobactersaccharolyticum; and Zymomonas, such as Zymomonas mobilis.

In a preferred aspect, the yeast is a Bretannomyces. In a more preferredaspect, the yeast is Bretannomyces clausenii. In another preferredaspect, the yeast is a Candida. In another more preferred aspect, theyeast is Candida sonorensis. In another more preferred aspect, the yeastis Candida boidinii. In another more preferred aspect, the yeast isCandida blankii. In another more preferred aspect, the yeast is Candidabrassicae. In another more preferred aspect, the yeast is Candidadiddensii. In another more preferred aspect, the yeast is Candidaentomophiliia. In another more preferred aspect, the yeast is Candidapseudotropicalis. In another more preferred aspect, the yeast is Candidascehatae. In another more preferred aspect, the yeast is Candida utilis.In another preferred aspect, the yeast is a Clavispora. In another morepreferred aspect, the yeast is Clavispora lusitaniae. In another morepreferred aspect, the yeast is Clavispora opuntiae. In another preferredaspect, the yeast is a Kluyveromyces. In another more preferred aspect,the yeast is Kluyveromyces fragilis. In another more preferred aspect,the yeast is Kluyveromyces marxianus. In another more preferred aspect,the yeast is Kluyveromyces thermotolerans. In another preferred aspect,the yeast is a Pachysolen. In another more preferred aspect, the yeastis Pachysolen tannophilus. In another preferred aspect, the yeast is aPichia. In another more preferred aspect, the yeast is a Pichiastipitis. In another preferred aspect, the yeast is a Saccharomyces spp.In another more preferred aspect, the yeast is Saccharomyces cerevisiae.In another more preferred aspect, the yeast is Saccharomyces distaticus.In another more preferred aspect, the yeast is Saccharomyces uvarum.

In a preferred aspect, the bacterium is a Bacillus. In a more preferredaspect, the bacterium is Bacillus coagulans. In another preferredaspect, the bacterium is a Clostridium. In another more preferredaspect, the bacterium is Clostridium acetobutylicum. In another morepreferred aspect, the bacterium is Clostridium phytofermentans. Inanother more preferred aspect, the bacterium is Clostridiumthermocellum. In another more preferred aspect, the bacterium isGeobacillus sp. In another more preferred aspect, the bacterium is aThermoanaerobacter. In another more preferred aspect, the bacterium isThermoanaerobacter saccharolyticum. In another preferred aspect, thebacterium is a Zymomonas. In another more preferred aspect, thebacterium is Zymomonas mobilis.

Commercially available yeast suitable for ethanol production include,e.g., BIOFERM™ AFT and XR (NABC—North American Bioproducts Corporation,GA, USA), ETHANOL RED™ yeast (Fermentis/Lesaffre, USA), FALI™(Fleischmann's Yeast, USA), FERMIOL™ (DSM Specialties), GERT STRAND™(Gert Strand AB, Sweden), and SUPERSTART™ and THERMOSACC™ fresh yeast(Ethanol Technology, WI, USA).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (co-fermentation) (Chen and Ho, 1993, Cloningand improving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TAL1 genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470;WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganismis Candida sonorensis. In another preferred aspect, the geneticallymodified fermenting microorganism is Escherichia col. In anotherpreferred aspect, the genetically modified fermenting microorganism isKlebsiella oxytoca. In another preferred aspect, the geneticallymodified fermenting microorganism is Kluyveromyces marxianus. In anotherpreferred aspect, the genetically modified fermenting microorganism isSaccharomyces cerevisiae. In another preferred aspect, the geneticallymodified fermenting microorganism is Zymomonas mobilis.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedcellulosic material or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, e.g., about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., e.g.,about 32° C. or 50° C., and about pH 3 to about pH 8, e.g., pH 4-5, 6,or 7.

In one aspect, the yeast and/or another microorganism are applied to thedegraded cellulosic material and the fermentation is performed for about12 to about 96 hours, such as typically 24-60 hours. In another aspect,the temperature is preferably between about 20° C. to about 60° C.,e.g., about 25° C. to about 50° C., about 32° C. to about 50° C., orabout 32° C. to about 50° C., and the pH is generally from about pH 3 toabout pH 7, e.g., about pH 4 to about pH 7. However, some fermentingorganisms, e.g., bacteria, have higher fermentation temperature optima.Yeast or another microorganism is preferably applied in amounts ofapproximately 105 to 10¹², preferably from approximately 10⁷ to 10¹⁰,especially approximately 2×10⁸ viable cell count per ml of fermentationbroth. Further guidance in respect of using yeast for fermentation canbe found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P.Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom1999), which is hereby incorporated by reference.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation products: A fermentation product can be any substancederived from the fermentation. The fermentation product can be, withoutlimitation, an alcohol (e.g., arabinitol, n-butanol, isobutanol,ethanol, glycerol, methanol, ethylene glycol, 1,3-propanediol [propyleneglycol], butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g.,pentane, hexane, heptane, octane, nonane, decane, undecane, anddodecane), a cycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane,and cyclooctane), an alkene (e.g. pentene, hexene, heptene, and octene);an amino acid (e.g., aspartic acid, glutamic acid, glycine, lysine,serine, and threonine); a gas (e.g., methane, hydrogen (H₂), carbondioxide (CO₂), and carbon monoxide (CO)); isoprene; a ketone (e.g.,acetone); an organic acid (e.g., acetic acid, acetonic acid, adipicacid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formicacid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid,glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid,malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid,succinic acid, and xylonic acid); and polyketide. The fermentationproduct can also be protein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. In a more preferred aspect, thealcohol is n-butanol. In another more preferred aspect, the alcohol isisobutanol. In another more preferred aspect, the alcohol is ethanol. Inanother more preferred aspect, the alcohol is methanol. In another morepreferred aspect, the alcohol is arabinitol. In another more preferredaspect, the alcohol is butanediol. In another more preferred aspect, thealcohol is ethylene glycol. In another more preferred aspect, thealcohol is glycerin. In another more preferred aspect, the alcohol isglycerol. In another more preferred aspect, the alcohol is1,3-propanediol. In another more preferred aspect, the alcohol issorbitol. In another more preferred aspect, the alcohol is xylitol. See,for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999,Ethanol production from renewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Silveira, M. M., and Jonas, R., 2002,The biotechnological production of sorbitol, Appl. Microbiol.Biotechnol. 59: 400-408; Nigam, P., and Singh, D., 1995, Processes forfermentative production of xylitol—a sugar substitute, ProcessBiochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi, N. and Blaschek, H.P., 2003, Production of acetone, butanol and ethanol by Clostridiumbeijerinckii BA101 and in situ recovery by gas stripping, World Journalof Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an alkane. Thealkane can be an unbranched or a branched alkane. In another morepreferred aspect, the alkane is pentane. In another more preferredaspect, the alkane is hexane. In another more preferred aspect, thealkane is heptane. In another more preferred aspect, the alkane isoctane. In another more preferred aspect, the alkane is nonane. Inanother more preferred aspect, the alkane is decane. In another morepreferred aspect, the alkane is undecane. In another more preferredaspect, the alkane is dodecane.

In another preferred aspect, the fermentation product is a cycloalkane.In another more preferred aspect, the cycloalkane is cyclopentane. Inanother more preferred aspect, the cycloalkane is cyclohexane. Inanother more preferred aspect, the cycloalkane is cycloheptane. Inanother more preferred aspect, the cycloalkane is cyclooctane.

In another preferred aspect, the fermentation product is an alkene. Thealkene can be an unbranched or a branched alkene. In another morepreferred aspect, the alkene is pentene. In another more preferredaspect, the alkene is hexene. In another more preferred aspect, thealkene is heptene. In another more preferred aspect, the alkene isoctene.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example, Richard, A., andMargaritis, A., 2004, Empirical modeling of batch fermentation kineticsfor poly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114,1997, Anaerobic digestion of biomass for methane production: A review.

In another preferred aspect, the fermentation product is isoprene.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. In another more preferred aspect,the ketone is acetone. See, for example, Qureshi and Blaschek, 2003,supra.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is polyketide.

Recovery. The fermentation product(s) can be optionally recovered fromthe fermentation medium using any method known in the art including, butnot limited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction.

For example, alcohol is separated from the fermented cellulosic materialand purified by conventional methods of distillation. Ethanol with apurity of up to about 96 vol. % can be obtained, which can be used as,for example, fuel ethanol, drinking ethanol, i.e., potable neutralspirits, or industrial ethanol.

Signal Peptide

The present invention also relates to an isolated polynucleotideencoding a signal peptide comprising or consisting of amino acids 1 to20 of SEQ ID NO: 2, amino acids 1 to 17 of SEQ ID NO: 4, amino acids 1to 23 of SEQ ID NO: 6, amino acids 1 to 16 of SEQ ID NO: 8, or aminoacids 1 to 19 of SEQ ID NO: 10. The polynucleotides may further comprisea gene encoding a protein, which is operably linked to the signalpeptide. The protein is preferably foreign to the signal peptide. In oneaspect, the polynucleotide for the signal peptide is nucleotides 1 to 60of SEQ ID NO: 1, nucleotides 1 to 51 of SEQ ID NO: 3, nucleotides 1 to69 of SEQ ID NO: 5, nucleotides 1 to 48 of SEQ ID NO: 7, or nucleotides1 to 57 of SEQ ID NO: 9.

The present invention also relates to nucleic acid constructs,expression vectors and recombinant host cells comprising suchpolynucleotides.

The present invention also relates to methods of producing a protein,comprising: (a) cultivating a recombinant host cell comprising suchpolynucleotide; and (b) recovering the protein

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andpolypeptides. The term “protein” also encompasses two or morepolypeptides combined to form the encoded product. The proteins alsoinclude hybrid polypeptides and fused polypeptides.

Preferably, the protein is a hormone, enzyme, receptor or portionthereof, antibody or portion thereof, or reporter. For example, theprotein may be a hydrolase, isomerase, ligase, lyase, oxidoreductase, ortransferase, e.g., an alpha-galactosidase, alpha-glucosidase,aminopeptidase, amylase, beta-galactosidase, beta-glucosidase,beta-xylosidase, carbohydrase, carboxypeptidase, catalase,cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, endoglucanase, esterase,glucoamylase, invertase, laccase, lipase, mannosidase, mutanase,oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Media

PDA plates were composed of 39 grams of potato dextrose agar anddeionized water to 1 liter.

YG agar plates were composed of 5.0 g of yeast extract, 10.0 g ofglucose, 20.0 g of agar, and deionized water to 1 liter.

YP medium was composed of 10 g of yeast extract, 20 g of Bactopeptone,and deionized water to 1 liter.

YPG medium was composed of 2% peptone, 1% yeast extract, and 2% glucosein deionized water.

LB medium was composed of 10 g of tryptone, 5 g of yeast extract, 5 g ofsodium chloride, and deionized water to 1 liter.

LB plates were composed of LB medium and 15 g of Bacto agar per liter ofLB medium.

NNCYP-PCS medium was composed of 5.0 g of NaNO₃, 3.0 g of NH₄Cl, 2.0 gof MES, 2.5 g of citric acid, 0.2 g of CaCl₂ 2H₂O, 1.0 g of BactoPeptone, 5.0 g of yeast extract, 0.2 g of MgSO₄ 7H₂O, 4.0 g of K₂HPO₄,1.0 ml of COVE trace elements solution, 2.5 g of glucose, 25.0 g ofpretreated corn stover (PCS), and deionized water to 1 liter.

COVE trace elements solution was composed of 0.04 g of Na₂B4O₇.10H₂O,0.4 g of CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g of MnSO₄.H₂O, 0.8 g ofNa₂MoO₂.2H₂O, 10 g of ZnSO₄.7H₂O, and deionized water to 1 liter.

Example 1: Cloning and Expression of an Aurantiporus alborubescens Aua2GH61 Polypeptide

Aurantiporus alborubescens strain NN008024 was isolated from a soil fromYunnan, China by directly plating the soil sample onto a PDA platefollowed by incubation at 37° C. for 5 days. The strain was thenpurified by transferring the mycelia onto an YG agar plate andidentified as Aurantiporus alborubescens based on both morphological andmolecular characterization (ITS sequencing).

The Aurantiporus alborubescens Aua2 GH61 polypeptide gene was cloned asdescribed below. Genomic DNA from Aurantiporus alborubescens strainNN008024 was isolated using a FASTDNA® SPIN Kit for Soil (MPBiomedicals, Solon, Ohio, USA) using a modification of themanufacturer's instructions. Briefly, the Kit was used with aFASTPREP®-24 Homogenization System (MP Biomedicals, Solon, Ohio, USA).A. alborubescens was grown in 5 ml of YP medium supplemented with 2%glucose for 48 hours at 30° C. Two ml of fungal material from thecultures were harvested by centrifugation at 14,000×g for 2 minutes. Thesupernatant was removed and the pellet resuspended in 500 μl ofdeionized water. The suspension was transferred to a Lysing Matrix Etube (FASTDNA® SPIN Kit) and 790 μl of sodium phosphate buffer and 100μl of MT buffer (FASTDNA® SPIN Kit) were added to the tube. The samplewas then secured in a FASTPREP™ System (MP Biomedicals, Solon, Ohio,USA) and processed for 60 seconds at a speed of 5.5 m/second. The samplewas then centrifuged at 14,000×g for two minutes and the supernatanttransferred to an EPPENDORF® tube. A 250 μl volume of PPS reagent fromthe FASTDNA® SPIN Kit was added and then the sample was mixed gently byinversion. The sample was again centrifuged at 14,000×g for 5 minutes.The supernatant was transferred to a 15 ml FALCON® 2059 tube. One ml ofBinding Matrix suspension (FASTDNA® SPIN Kit) was added and then mixedby inversion for two minutes. The sample was placed in a stationary tuberack and the Binding Matrix was allowed to settle for 3 minutes. Then500 μl of the supernatant were removed and discarded and the remainingsample was resuspended in the Binding Matrix. This sample was thentransferred to a SPIN™ filter (FASTDNA® SPIN Kit) and centrifuged at14,000×g for 1 minute. The catch tube was emptied and the remainingmatrix suspension added to the SPIN™ filter. The sample was againcentrifuged at 14,000×g for 1 minute. A 500 μl volume of SEWS-M solution(FASTDNA® SPIN Kit) was added to the SPIN™ filter and the sample wascentrifuged at the same speed for 1 minute. The catch tube was emptiedand the SPIN™ filter replaced in the catch tube. The unit wascentrifuged at 14,000×g for 2 minutes to dry the matrix of residualSEWS-M wash solution. The SPIN™ filter was placed in a fresh catch tubeand allowed to air dry for 5 minutes at room temperature. The matrix wasgently resuspended in 100 μl of DES (FASTDNA® SPIN Kit) with a pipettip. The unit was centrifuged at 14,000×g for 1 minute. Theconcentration of the DNA harvested from the catch tube was determined at260 nm.

The Aurantiporus alborubescens Aua2 GH61 polypeptide gene was clonedusing the primers shown below. The PCR primers were designed to amplifythe entire open reading frame from the ATG start codon through thetermination codon. The primers were synthesized with 15 base pair 5′sequences homologous to the border of the Eco RI-Not I cloning site ofplasmid pXYG1051 (WO 2005/080559).

Primer Aua2-RI: (SEQ ID NO: 11) 5'-GCGGAATTCAACATGCGAACCATCGCCA-3'Primer Aua2-NotI: (SEQ ID NO: 12) 5'-ATATGCGGCCGCATAAGCAACTCCCTCAGAG-3'Bold letters represent A. alborubescens Aua2 GH61 polypeptide codingsequence. The underlined sequence contains the Eco RI restriction siteon the forward primer (Aua2-RI) and the Not I restriction site on thereverse primer (Aua2-NotI). When the primers are used in a PCR reactionwith cDNA or genomic DNA from A. alborubescens, a fragment can beproduced that can be restricted with Eco RI and Not I to produce anotherfragment that can be cloned directionally into a suitable vector withthe same restriction sites.

The PCR reaction (50 μl) was composed of 25 μl of 2× IPROOF™ HF MasterMix (Bio-Rad Laboratories, Inc., Hercules, Calif., USA), 1 μl of primerAua2-RI (100 μM), 1 μl of primer Aua2-NotI (100 μM), 1 μl of A.alborubescens genomic DNA (200 ng/μl), and 22 μl of deionized water. TheIPROOF™ HF Master Mix contains buffer, dNTPs, and a thermostable DNApolymerase blend. The PCR reaction was incubated in a DYAD® Dual-BlockThermal Cycler (MJ Research, Waltham, Mass., USA) programmed for 1 cycleat 98° C. for 60 seconds; 30 cycles each at 98° C. for 10 seconds, 50°C. for 30 seconds, and 72° C. for 60 seconds; and 1 cycle at 72° C. for10 minutes. Samples were cooled to 10° C. before removal and furtherprocessing.

Five μl of the PCR reaction were analyzed by 1% agarose gelelectrophoresis using 40 mM Tris base-20 mM sodium acetate-1 mM disodiumEDTA (TAE) buffer where an approximately 1.1 kb product band wasobserved. The remaining PCR reaction was purified using an ILLUSTRA™GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare,Buckinghamshire, UK) according to the manufacturer's instructions.

The methodology for cloning the A. alborubescens Aua2 GH61 polypeptideencoding sequence into a suitable expression vector and transformationof the vector into Aspergillus oryzae and selection of Aspergillustransformants producing the GH61 polypeptide is described in Example 2of WO 2005/080559. Briefly, the purified PCR product was ligated intothe Aspergillus expression vector pXYG1051 (WO 2005/080559), which is aderivative of pMSTr46 (WO 2003/070956) modified as described in WO2005/080559. Plasmid pXYG1051 was digested with Eco RI-Not I. The 1.1 kbgene fragment and the digested vector were ligated together in areaction (10.2 μl) composed of 1 μl of Eco RI-Not I digested pXYG1051(10 ng/μl of 10 mM Tris, 1 mM EDTA pH 7.5 [TE]), 8 μl of the Aua2 PCRfragment (30 ng/μl), 1 μl of 10× T4 DNA ligase buffer (Promega Corp.,Madison, Wis., USA), and 0.2 μl of T4 DNA ligase (Promega Corp.,Madison, Wis., USA). The reaction was incubated overnight at 16° C.

A 1 μl volume of the ligation reaction mixture was transformed into ONESHOT® TOP10 Chemically Competent E. coli cells (50 μl; Invitrogen,Carlsbad, Calif., USA) according to the manufacturer's instructions. Thetransformation was plated onto LB agar plates supplemented with 100 μgof ampicillin per ml and the plates were incubated overnight at 37° C.Six colonies were chosen from several hundred that grew under selectionand inoculated into 2 ml of LB medium supplemented with 100 μg ofampicillin per ml in FALCON® tubes. Plasmid DNA was isolated using aQIAprep Spin Miniprep Kit (Qiagen GmBH, Hilden Germany) according to themanufacturer's instructions. The plasmid DNA was digested with Eco RIand Hind III and the digests analyzed by 1% agarose gel electrophoresisusing 90 mM Tris-borate and 1 mM EDTA (TBE) buffer, which indicated thatall six clones contained an insert of the correct size of 1.5 kb. Theclones were then sequenced using an ABI 3730 XL Genetic Analyzer(Applied Biosystems, Foster City, Calif., USA). One error free clonedesignated E. coli NP001127-5 comprising the A. alborubescens Aua2 GH61polypeptide genomic DNA sequence of SEQ ID NO: 1 was selected.

E. coli NP001127-5 was cultivated in 50 ml of LB medium supplementedwith 100 μg of ampicillin per ml. Plasmid DNA was isolated and purifiedusing a Plasmid Midi Kit (Qiagen GmBH, Hilden Germany) according to themanufacturer's instructions. A quantity of 1.6 μg of Aua2 GH61 plasmidDNA was used to transform Aspergillus oryzae JaL355 (WO 2001/98484)protoplasts prepared according to the method of EP0238023 B1, (pages14-15) for A. oryzae MT3568 protoplasts. Transformants were re-isolatedtwice under selective and non-inducing conditions on COVE minimal plates(Cove, 1966, Biochim. Biophys. Acta 133: 51-56) with 1M sucrose as acarbon source and 10 mM nitrate. To test expression of the Aau2 GH61polypeptide, 18 transformants were grown for 3 days and 4 days at 30° C.in tubes with 10 ml of YPG medium. Supernatants were analyzed bySDS-PAGE using NUPAGE® 10% Bis-Tris SDS gels (Invitrogen, Carlsbad,Calif., USA) according to the manufacturer. All Aspergillus isolatesgrew well in YPG medium when induced for expression of the Aau2 GH61polypeptide. One Aspergillus oryzae transformant producing the Aau2 GH61polypeptide, as judged by SDS-PAGE analysis, was chosen for further workand designated A. oryzae EXP3192. A. oryzae EXP3192 was fermented in1000 ml Erlenmeyer shake flasks with 100 ml of YP medium supplementedwith 2% glucose at 26° C. for 4 days with agitation at 85 rpm. Severalshake flasks were used to provide enough culture broth for subsequentfiltration, concentration, and/or purification of the recombinantlyproduced polypeptide.

An alternative method for cloning and expressing the Aua2 GH61polypeptide is described below. Based on the nucleotide sequence of SEQID NO: 1, a synthetic gene can be obtained from a number of vendors suchas Gene Art (GENEART AG, Regensburg, Germany) or DNA 2.0 (DNA2.0, MenloPark, Calif., USA). The synthetic gene can be designed to incorporateadditional DNA sequences such as restriction sites or homologousrecombination regions to facilitate cloning into an expression vector.Using the two synthetic oligonucleotide primers Aua2-RI and Aua2-NotIdescribed above, a simple PCR reaction can be used to amplify thefull-length open reading frame from the synthetic gene of SEQ ID NO: 1.The gene can then be cloned into an expression vector, for example, asdescribed above and expressed in a host cell, for example, Aspergillusoryzae. The GH61 polypeptide expressed in this way corresponds to SEQ IDNO: 2.

Example 2: Characterization of the Aurantiporus alborubescens Aua2 GH61Polypeptide

The genomic DNA sequence and deduced amino acid sequence of theAurantiporus alborubescens Aua2 GH61 polypeptide encoding sequence areshown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The genomic DNAsequence contains 8 introns of 55 bp (nucleotides 120 to 174), 57 bp(nucleotides 232 to 288), 52 bp (nucleotides 437 to 488), 56 bp(nucleotides 555 to 610), 48 bp (nucleotides 782 to 829), 123 bp(nucleotides 933 to 1055), 55 bp (nucleotides 1151 to 1205), and 53 bp(nucleotides 1393 to 1445). The genomic DNA fragment encodes apolypeptide of 322 amino acids. The % G+C content of the polypeptideencoding sequence is 55.6%. Using the SignalP software program (Nielsenet al., 1997, Protein Engineering 10: 1-6), a signal peptide of 20residues was predicted. The SignalP prediction is in accord with thenecessity for having a histidine reside at the N-terminus in order forproper metal binding and hence protein function to occur (See Harris etal., 2010, Biochemistry 49: 3305, and Quinlan et al., 2011, Proc. Natl.Acad. Sci. USA 108: 15079). The predicted mature protein contains 302amino acids with a predicted molecular mass of 31 kDa and a predictedisoelectric point of 6.64. The protein contains a carbohydrate bindingmodule of the CBM1 type at the C terminus (amino acids 288 to 322 of SEQID NO: 2).

A comparative alignment of mature Family 61 amino acid sequences,without the signal peptides, was determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of EMBOSS with a gap open penalty of10, a gap extension penalty of 0.5, and the EBLOSUM62 matrix. Thealignment showed that the deduced amino acid sequence of theAurantiporus alborubescens Aua2 GH61 mature polypeptide shares 76.83%identity (excluding gaps) to the deduced amino acid sequence of a GH61polypeptide from Moniliophthora perniciosa (SWISSPROT E2LXA6).

Example 3: Effect of Aurantiporus alborubescens Aua2 GH61 Polypeptide onHydrolysis of Pretreated Corn Stover

Culture broth prepared as described in Example 1 was concentratedapproximately 20-fold using an Amicon ultrafiltration device (Millipore,Bedford, Mass., USA, 10 kDa polyethersulfone membrane, 40 psi, 4° C.).Protein concentration was determined using a BCA Protein Assay (BCAProtein Assay Kit; Thermo Fisher Scientific, Waltham, Mass., USA). Cornstover was pretreated and prepared as an assay substrate as described inWO 2005/074647 to generate pretreated corn stover (PCS). The basecellulase mixture used to assay enhancing activity was prepared fromTrichoderma reesei strain SMA135 (WO 2008/057637).

Hydrolysis of PCS was conducted using 1.6 ml deep-well plates (Axygen,Santa Clara, Calif., USA) with a total reaction volume of 1.0 ml and aPCS concentration of 50 mg/ml in 1 mM manganese sulfate-50 mM sodiumacetate pH 5.0. The Aurantiporus alborubescens Aua2 GH61 polypeptide wasseparately added to the base cellulase mixture at concentrations rangingfrom 0 to 100% of the protein concentration of the base cellulasemixture. Incubation was at 50° C. for 72 hours. Assays were performed intriplicate. Aliquots were centrifuged, and the supernatant liquid wasfiltered by centrifugation (MULTISCREEN® HV 0.45 μm, Millipore,Billerica, Mass., USA) at 3000 rpm for 10 minutes using a platecentrifuge (SORVALL® RT7, Thermo Fisher Scientific, Waltham, Mass.,USA). When not used immediately, filtered hydrolysate aliquots werefrozen at −20° C. Sugar concentrations of samples diluted in 0.005MH₂SO₄ with 0.05% w/w benzoic acid were measured after elution by 0.005MH₂SO₄ with 0.05% w/w benzoic acid at a flow rate of 0.6 ml/minute from a4.6×250 mm AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules,Calif., USA) at 65° C. with quantitation by integration of glucose andcellobiose signals from refractive index detection (CHEMSTATION®,AGILENT® 1100 HPLC, Agilent Technologies, Santa Clara, Calif., USA)calibrated by pure sugar samples (Absolute Standards Inc., Hamden,Conn., USA). The resultant equivalents were used to calculate thepercentage of cellulose conversion for each reaction. The degree ofcellulose conversion to glucose plus cellobiose sugars (conversion, %)was calculated using the following equation:

Conversion(%)=(glucose+cellobiose×1.053)(mg/ml)×100×162/(Cellulose(mg/ml)×180)=(glucose+cellobiose×1.053)(mg/ml)×100/(Cellulose(mg/ml)×1.111)

In this equation the factor 1.111 reflects the weight gain in convertingcellulose to glucose, and the factor 1.053 reflects the weight gain inconverting cellobiose to glucose. Cellulose in PCS was determined by alimit digest of PCS to release glucose and cellobiose.

The result of adding increasing amounts of the Aurantiporusalborubescens Aua2GH61 polypeptide to the base cellulase mix are shownin FIG. 1. Addition of the Aurantiporus alborubescens Aua2 GH61polypeptide provided a stimulation factor of 1.17 at a 100% additionlevel.

Example 4: Cloning and Expression of an Aurantiporus alborubescens Aua1GH61 Polypeptide

The Aurantiporus alborubescens Aua1 GH61 polypeptide gene was clonedfrom a cDNA library using the primers shown below for cloning intoplasmid pDau109 (WO 2005/042735). The PCR primers were designed toamplify the entire open reading frame from the ATG start codon until thetermination codon. The primers were synthesized with 15 base pair 5′sequences homologous to the border of the Hind III-Bam HI cloning siteof pDau109.

Primer F-Aua1: (SEQ ID NO: 13)5'-ACACAACTGGGGATCCACCATGAAGGCTATCTTGGCTATTT-3' Primer R-Aua1:(SEQ ID NO: 14) 5'-AGATCTCGAGAAGCTTAACCACGCCACACAGCAGG-3'Bold letters represent A. alborubescens Aua1 GH61 polypeptide codingsequence. The underlined sequence contains the Bam HI restriction siteon the forward primer (F-Aua1) and the Hind III restriction site on thereverse primer (R-Aua1). When the primers are used in a PCR reactionwith cDNA or genomic DNA from A. alborubescens, a fragment can beproduced that can be restricted with Bam HI and Hind III to produceanother fragment that can be cloned directionally into a suitable vectorwith the same restriction sites. Additionally, the unrestricted fragmentcan be used for recombinational cloning into pDau109 using an IN-FUSION™PCR Cloning Kit (Clontech Laboratories, Inc., Mountain View, Calif.,USA).

A cDNA library was generated from A. alborubescens strain NN008024according to the following protocol. A. alborubesens was inoculated into1000 ml Erlenmeyer shake flasks containing 100 ml of NNCYP+PCS mediumand incubated at 26° C. for 4 days with agitation at 85 rpm. The fungalmycelia were harvested by filtration through MIRACLOTH® (Calbiochem, SanDiego, Calif., USA) before being frozen in liquid nitrogen. The myceliawere then pulverized into a powder by milling the frozen myceliatogether with an equal volume of dry ice in a KRUPS® KM 75 coffeegrinder (Krups, Shelton, Conn., USA) precooled with liquid nitrogen. Thepowder was transferred into a liquid nitrogen prechilled mortar andpestle (previously baked at 250° C. for 12 hours) and ground to a finepowder with a small amount of baked quartz sand (baked at 250° C. for 12hours). The powdered mycelial material was kept at −80° C. until use.Total RNA was extracted according to a modified TRIZOL® method adaptedfor total RNA extraction for fungi. Briefly, to each of six 2 mlEPPENDORF® tubes, 800 μl of TRIZOL® (Life Technologies, Carlsbad,Calif., USA) was added. The powdered mycelia were distributed evenlywith a metal spoon (baked at 250° C. for 12 hours) to the EPPENDORF®tubes in a quantity that did not exceed the total 2 ml volume of thetube. The samples were incubated in a 50° C. water bath for 5 minutesand then 200 μl of RNase-free chloroform was added. The samples werevortexed vigorously for 20 seconds and allowed to stand at roomtemperature for 10 minutes. The samples were centrifuged at 12,000×g for10 minutes at room temperature and the top phase was then decanted to anew tube in which an equal volume of phenol-chloroform mix (SigmaChemical, Co., St. Louis, Mo., USA) was added. The samples werecentrifuged at 12,000×g for 10 minutes. The top phase was transferred toa new tube and an equal volume of chloroform-isoamyl alcohol (24:1 v/v)was added. The samples were again centrifuged at 12,000×g for 10minutes. The aqueous phase was transferred to a new tube and 250 μl ofRNase-free isopropanol (Fluka, Milwaukee, Wis., USA) were added and thesamples mixed after which they were incubated at room temperature for 15minutes. The samples were centrifuged at 20,000×g for 15 minutes at 4°C. The supernatants were carefully removed and 700 μl of RNase-free 70%ethanol were added to each of the RNA pellets, which were thencentrifuged at 20,000×g for 5 minutes at 4° C. The supernatants werecarefully removed and the RNA pellets air dried. The RNA pellets wereresuspended in 300 μl of diethylpyrocarbonate (DEPC)-treated water. Thesamples were heated at 65° C. for 10 minutes to aid in resuspension. Thesix dissolved RNA samples were all pooled into one tube and then ethanolprecipitated. Briefly, 1/10 volume of RNase-free 3M sodium acetate pH5.2 was added followed by 2 volumes of RNase-free 96% ethanol. Thesample was mixed and left to precipitate overnight at −20 C. The samplewas then centrifuged at 20,000×g for 30 minutes at 4° C. The supernatantwas removed and the pellet washed with 250 μl of RNase-free 70% ethanol.The supernatant was carefully removed and the pellet allowed to air dry.The RNA pellet was then resuspended in 300 μl of DEPC-treated water andstored at −80° C. until use.

PolyA enriched RNA was made from the total RNA and isolated using anmTRAP™ Maxi mRNA Purification Kit (ActiveMotif Inc., Carlsbad, Calif.,USA) according to the manufacturer's instructions.

The cDNA library was constructed with a SMART™ cDNA Library ConstructionKit (Clontech Laboratories, Inc., Mountain View, Calif., USA) accordingto the manufacturer's instructions. The cDNA was size selected with amolecular weight cut-off of 500 base pairs by agarose gelelectrophoresis. The plasmid pMHas10 (SEQ ID NO: 11) was used instead ofthe phage vector supplied with the Kit. Plasmid pMHas10 was prepared byrestricting 5 μg of the plasmid with Sfi I and isolating the plasmidfrom the stuffer insert by 1% agarose gel electrophoresis using TAEbuffer. Plasmid DNA was isolated from a pool of 50,000 colonies, scrapedfrom LB plates supplemented with 50 μg of kanamycin per ml. Plasmid DNAfrom the pooled bacteria was used for preparation of a plasmid libraryusing the JETSTAR® 2.0 Plasmid Mini/Midi/Maxi-Protocol (GenoMed GmbH,Löhne, Germany).

The cDNA library was diluted to 100 ng/μl in MilliQ water and used astemplate for a PCR reaction using the Aua1 primers. The conditions wereidentical to those used for producing the PCR fragment in Example 1except that primer F-Aua1 and primer R-Aua1 were used.

Five μl of the PCR product were analyzed by 1% agarose electrophoresisusing TAE buffer, which showed the presence of a single band with thepredicted size of 740 bp. The remaining PCR reaction was purified usingan ILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit. An IN-FUSION™PCR Cloning Kit was used for cloning the fragments into plasmid pDau109.Two pg of pDau109 was digested with Bam HI and Hind III and the digestedplasmid was run on a 1% agarose gel using TBE buffer in order to removethe stuffer fragment from the restricted plasmid. The bands werevisualized by the addition of SYBR® Safe DNA gel stain (Invitrogen Inc.,Carlsbad, Calif., USA) into the agarose gel and use of a 470 nmwavelength transilluminator. The band corresponding to the restrictedplasmid was excised from the gel and purified using an ILLUSTRA™ GFX™PCR DNA and Gel Band Purification Kit. The plasmid was eluted into 10 mMTris pH 8.0 and its concentration adjusted to 20 ng/μl. Using theIN-FUSION™ PCR Cloning Kit the 750 bp PCR fragment (50 ng) was clonedinto plasmid pDau109 (20 ng) digested with Bam HI and Hind III. Thereaction was transformed into FUSION-BLUE™ E. coli cells (ClontechLaboratories, Inc., Mountain View, Calif., USA) according to themanufacturer's protocol and plated onto LB agar plates supplemented with50 μg of ampicillin per ml. After incubation overnight at 37° C.,colonies were observed growing under selection on the LB ampicillinplates. Ten colonies transformed with the Aau1 GH61 construct werecultivated in LB medium supplemented with 50 μg of ampicillin per ml andplasmid was isolated using a JETQUICK™ Plasmid Purification Spin Kit(GenoMed GmbH, Löhne, Germany) according to the manufacturer'sinstructions.

Isolated plasmids were sequenced with vector primers in order todetermine a representative plasmid expression clone that was free of PCRerrors. One error free Aau1 GH61 clone comprising SEQ ID NO: 3 wasselected for further work. Plasmid DNA was then isolated using aJETQUICK™ 2.0 Plasmid Mini/Midi/Maxi-Protocol (GenoMed GmbH, Löhne,Germany). Transformation of the selected plasmid into Aspergillus oryzaeJaL355 was performed as described in Example 1. One Aspergillus oryzaetransformant producing acceptable levels of the Aau1 GH61 polypeptide,as judged by SDS-PAGE analysis (Example 1), was chosen for further workand designated A. oryzae EXP3380. A. oryzae EXP3380 strain was fermentedin 1000 ml Erlenmeyer shake flasks with 100 ml of YP medium supplementedwith 2% glucose at 26° C. for 4 days with agitation at 85 rpm. Severalshake flasks were used to provide enough culture broth for subsequentfiltration, concentration, and/or purification of the recombinantlyproduced polypeptide.

An alternative method for cloning and expressing the Aua1 GH61 gene isdescribed below. Based on the nucleotide sequence of SEQ ID NO: 3, asynthetic gene can be obtained from a number of vendors such as Gene Artor DNA 2.0. The synthetic gene can be designed to incorporate additionalDNA sequences such as restriction sites or homologous recombinationregions to facilitate cloning into an expression vector. Using the twosynthetic oligonucleotide primers Aua2-RI and Aua2-NotI described above,a simple PCR reaction can be used to amplify the full-length openreading frame from the synthetic gene of SEQ ID NO: 3. The gene can thenbe cloned into an expression vector, for example, as described inExample 1 and expressed in a host cell, for example, Aspergillus oryzae.The GH61 polypeptide expressed in this way corresponds to SEQ ID NO: 4.

Example 5: Characterization of the Aurantiporus alborubescens Aua1 GH61Polypeptide

The cDNA sequence and deduced amino acid sequence of the A.alborubescens Aua1 GH61 polypeptide encoding sequence are shown in SEQID NO: 3 and SEQ ID NO: 4, respectively. The cDNA fragment encodes apolypeptide of 234 amino acids. The % G+C content of the polypeptideencoding sequence is 56%. Using the SignalP software program (Nielsen etal., 1997, supra), a signal peptide of 17 residues was predicted. TheSignalP prediction is in accord with the necessity for having ahistidine reside at the N-terminus in order for proper metal binding andhence protein function to occur (See Harris et al., 2010, supra, andQuinlan et al., 2011, supra). The predicted mature protein contains 217amino acids with a predicted molecular mass of 23 kDa and an isoelectricpoint of 5.97.

A comparative alignment of mature Family 61 amino acid sequences,without the signal peptides, was determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of EMBOSS with gap open penalty of 10, gap extensionpenalty of 0.5, and the EBLOSUM62 matrix. The alignment showed that thededuced amino acid sequence of the A. alborubescens Aua1 GH61 maturepolypeptide shares 62.09% identity (excluding gaps) to the deduced aminoacid sequence of a GH61 polypeptide from Schizophyllum commune H4-8(SWISSPROT D8QHH2).

Example 6: Effect of Aurantiporus alborubescens Aua1 GH61 Polypeptide onHydrolysis of Pretreated Corn Stover

Culture broth was prepared as described in Example 4 and concentratedapproximately 20-fold as described in Example 3. PCS hydrolysisexperiments and determination of the degree of cellulose conversion wasperformed according to the procedures described in Example 3.

The result of adding increasing amounts of the Aurantiporusalborubescens Aua1 GH61 polypeptide to the base cellulase mix are shownin FIG. 2. Addition of the Aurantiporus alborubescens Aua1 GH61polypeptide provided a stimulation factor of 1.17 at a 100% additionlevel.

Example 7: Cloning and Expression of a Trichophaea saccata Tsa1 GH61Polypeptide

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Trichophaea saccata CBS 804.70 GH61 gene from a cDNA libraryprepared according to WO 2010/088387. The PCR primers were designed toamplify the entire open reading frame from the ATG start codon until thetermination codon. The primers were synthesized with 15 base pair 5′sequences homologous to the border of the cloning site for Hind III-BamHI digested pDau109.

Primer F-Tsa1: (SEQ ID NO: 15)5'-ACACAACTGGGGATCCACCATGACGCCCCTGAAACTCC-3' Primer R-Tsa1:(SEQ ID NO: 16) 5'-AGATCTCGAGAAGCTTACTTACCGGTCCAAACCGGT-3'

Bold letters represent Trichophaea saccata Tsa1 GH61 polypeptide codingsequence. Restriction sites are underlined. The remaining sequence ishomologous to the insertion sites of pDau109.

The PCR reaction (40 μl) was composed of 20 μl of 2× IPROOF™ HF MasterMix, 1 μl of primer F-Tsa1 (100 μM), 1 μl of primer R-Tsa1 (100 μM), 1μl of cDNA (100 ng/μl), and 17 μl of deionized water. The PCR reactionwas incubated in a DYAD® Dual-Block Thermal Cycler programmed for 1cycle at 98° C. for 60 seconds; 30 cycles each at 98° C. for 10 seconds,55° C. for 10 seconds, and 72° C. for 60 seconds; and 1 cycle at 72° C.for 10 minutes. Samples were cooled to 10° C. before removal and furtherprocessing.

Five μl of the PCR reaction were analyzed by 1% agarose gelelectrophoresis using TAE buffer where an approximately 750 bp productband was observed. The remaining PCR reaction was purified using anILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

Plasmid pDau109 (2 μg) was digested with the restriction enzymes Bam HIand Hind III and the digested plasmid was run on a 1% agarose gel usingTBE buffer in order to remove the stuffer fragment from the restrictedplasmid. The bands were visualized by the addition of SYBR® Safe DNA gelstain into the agarose gel and use of a 470 nm wavelengthtransilluminator. The band corresponding to the restricted plasmid wasexcised and purified using an ILLUSTRA™ GFX™ PCR DNA and Gel BandPurification Kit. The plasmid was eluted into 10 mM Tris pH 8.0 and itsconcentration adjusted to 20 ng/μl. An IN-FUSION™ PCR Cloning Kit wasused to clone the 750 bp PCR fragment (50 ng) into pDau109 digested withBam HI and Hind III (20 ng). The IN-FUSION™ reaction was transformedinto FUSION-BLUE™ E. coli cells according to the manufacturer's protocoland plated onto LB agar plates supplemented with 50 μg of ampicillin perml. After incubation overnight at 37° C., colonies were observed growingunder selection on the LB ampicillin plates. Ten transformants wereselected at random and cultivated in LB medium supplemented with 50 μgof ampicillin per ml. Plasmid DNA was isolated using a JETQUICK™ PlasmidPurification Spin Kit according to the manufacturer's instructions.

Isolated plasmids were sequenced with vector primers in order todetermine a representative plasmid expression clone that was free of PCRerrors. One error-free Tsa1 GH61 clone was selected. Plasmid DNA wasthen isolated using the JETSTAR® 2.0 Plasmid Mini/Midi/Maxi-Protocol.The purified plasmid DNA was transformed into Aspergillus oryzae Bech2according to the method described in WO 2005/042735, pages 34-35.Aspergillus transformants were grown and then analyzed for production ofTsa1 GH61 protein by SDS-PAGE analysis according to Example 1. Two bandswere observed for all transformants analyzed, a 23 kDa band and a 27 kDaband. The larger band may be the result of glycosylation. OneAspergillus oryzae transformant producing the Tsa1 GH61 polypeptide, asjudged by SDS-PAGE analysis, was chosen for further work and designatedA. oryzae EXP3315. A. oryzae EXP3315 was fermented in 1000 ml Erlenmeyershake flasks with 100 ml of YP medium supplemented with 2% glucose at26° C. for 4 days with agitation at 85 rpm. Several shake flasks wereused to provide enough culture broth for subsequent filtration,concentration and/or purification of the recombinantly producedpolypeptide.

An alternative method for cloning and expressing the Tsa1 GH61polypeptide is described below. Based on the nucleotide sequence of SEQID NO: 5, a synthetic gene can be obtained from a number of vendors suchas Gene Art or DNA 2.0. The synthetic gene can be designed toincorporate additional DNA sequences such as restriction sites orhomologous recombination regions to facilitate cloning into anexpression vector. Using the two synthetic oligonucleotide primersF-Tsa1 and R-Tsa1 described above, a simple PCR reaction can be used toamplify the full-length open reading frame from the synthetic gene ofSEQ ID NO: 5. The gene can then be cloned into an expression vector, forexample, as described above and expressed in a host cell, for example,Aspergillus oryzae. The GH61 polypeptide expressed in this waycorresponds to SEQ ID NO: 6.

Example 8: Characterization of the Trichophaea saccata Tsa1 GH61Polypeptide

The cDNA sequence and deduced amino acid sequence of the Trichophaeasaccata CBS 804.70 Tsa1 GH61 polypeptide encoding sequence are shown inSEQ ID NO: 5 and SEQ ID NO: 6, respectively. The cDNA fragment encodes apolypeptide of 233 amino acids. The G+C content of the polypeptideencoding sequence is 59%. Using the SignalP software program (Nielsen etal., 1997, supra), a signal peptide of 23 residues was predicted. TheSignalP prediction is in accord with the necessity for having ahistidine reside at the N-terminus in order for proper metal binding andhence protein function to occur (See Harris et al., 2010, supra, andQuinlan et al., 2011, supra). The predicted mature protein contains 210amino acids with a predicted molecular mass of 23 kDa and a predictedisoelectric point of 5.08.

A comparative alignment of mature Family 61 amino acid sequences,without the signal peptides, was determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of EMBOSS with a gap open penalty of 10, a gap extensionpenalty of 0.5, and the EBLOSUM62 matrix. The alignment showed that thededuced amino acid sequence of the Trichophaea saccata Tsa1 GH61 maturepolypeptide shares 57.21% identity (excluding gaps) to the deduced aminoacid sequence of a GH61 polypeptide from Aspergillus terreus (SWISSPROTQ0CJQ7).

Example 9: Effect of Trichophaea saccata Tsa1 GH61 Polypeptide onHydrolysis of Pretreated Corn Stover

Culture broth was prepared as described in Example 7 and concentratedapproximately 20-fold as described in Example 3. PCS hydrolysisexperiments and determination of the degree of cellulose conversion wasperformed according to the procedures described in Example 3.

The result of adding increasing amounts of the Trichophaea saccata Tsa1GH61 polypeptide to the base cellulase mix are shown in FIG. 3. Additionof the Trichophaea saccata Tsa1 GH61 polypeptide provided a stimulationfactor of 1.31 at a 100% addition level.

Example 10: Cloning and Expression of a Trichophaea saccata Tsa2 GH61Polypeptide

The Trichophaea saccata CBS 804.70 Tsa2 GH61 polypeptide gene was clonedusing the cDNA library obtained according to Example 7 and the primersshown below for cloning into pDau109. The PCR primers were designed toamplify the entire open reading frame from the ATG start codon throughthe termination codon. The primers were synthesized with 15 base pair 5′sequences homologous to the border of the Hind III-Bam HI cloning siteof pDau109.

Primer F-Tsa2: (SEQ ID NO: 17)5'-ACACAACTGGGGATCCACCATGAAATGCCTTCTCTCCCT-3' Primer R-Tsa2: (SEQ ID NO: 18) 5'-AGATCTCGAGAAGCTTAGCATGTAAACGGCCTTGGG-3'

Bold letters represent T. saccata Tsa2 GH61 polypeptide coding sequence.Restriction site are underlined. The remaining sequence is homologous tothe insertion sites of pDau109.

The PCR reaction (40 μl) was composed of 20 μl of 2× IPROOF™ HF MasterMix, 1 μl of primer F-Tsa2 (100 μM), 1 μl of primer R-Tsa2 (100 μM), 1μl of cDNA (100 ng/μl), and 17 μl of deionized water. The PCR reactionwas incubated in a DYAD® Dual-Block Thermal Cycler programmed for 1cycle at 98° C. for 60 seconds; 30 cycles each at 98° C. for 10 seconds,55° C. for 10 seconds, and 72° C. for 60 seconds; and 1 cycle at 72° C.for 10 minutes. Samples were cooled to 10° C. before removal and furtherprocessing.

Five μl of the PCR reaction were analyzed by 1% agarose gelelectrophoresis using TAE buffer where an approximately 735 bp productband was observed. The remaining PCR reaction was purified using anILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

An IN-FUSION™ PCR Cloning Kit was used for cloning the 735 bp PCRfragment into the vector pDau109 prepared as described in Example 4. TheIN-FUSION™ cloning was performed according to the manufacturer'sinstructions and Example 4 for pDau109 and the Tsa2 insert. TheIN-FUSION™ reaction was then transformed into FUSION-BLUE™ E. coli cellsaccording to the manufacturer's protocol and plated onto LB agar platessupplemented with 50 μg of ampicillin per ml. After incubation overnightat 37° C., colonies were observed growing under selection. Tentransformants were selected at random and cultivated in LB mediumsupplemented with 50 μg of ampicillin per ml. Plasmid DNA was isolatedusing a JETQUICK™ Plasmid Purification Spin Kit according to themanufacturer's instructions.

Isolated plasmids were sequenced with vector primers in order todetermine a representative plasmid expression clone that was free of PCRerrors. One error-free Tsa2 GH61 clone was selected. Plasmid DNA wasthen isolated using the JETSTAR® 2.0 Plasmid Mini/Midi/Maxi-Protocol.The purified plasmid DNA was transformed into Aspergillus oryzae Bech2according to the method described in WO 2005/042735, pages 34-35. Thetransformants were grown and their culture broths analyzed as describedin Example 1. For transformants producing recombinant protein, a proteinband of 23 kDa was observed. One Aspergillus oryzae transformantproducing acceptable levels of the Tsa2 GH61 polypeptide, as judged bySDS-PAGE analysis, was chosen for further work and designated A. oryzaeEXP3316. A. oryzae EXP3316 was fermented as described in Example 7 toprovide enough culture broth for subsequent filtration, concentration,and/or purification of the recombinantly produced polypeptide.

An alternative method for cloning and expressing the Tsa2 GH61polypeptide is described below. Based on the nucleotide sequence of SEQID NO: 7, a synthetic gene can be obtained from a number of vendors suchas Gene Art or DNA 2.0. The synthetic gene can be designed toincorporate additional DNA sequences such as restriction sites orhomologous recombination regions to facilitate cloning into anexpression vector. Using the two synthetic oligonucleotide primersF-Tsa2 and R-Tsa2 described above, a simple PCR reaction can be used toamplify the full-length open reading frame from the synthetic gene ofSEQ ID NO: 7. The gene can then be cloned into an expression vector, forexample, as described above and expressed in a host cell, for example,Aspergillus oryzae. The GH61 polypeptide expressed in this waycorresponds to SEQ ID NO: 8.

Example 11: Characterization of the Trichophaea saccata Tsa2 GH61Polypeptide

The cDNA sequence and deduced amino acid sequence of the Trichophaeasaccata Tsa2 GH61 polypeptide encoding sequence are shown in SEQ ID NO:7 and SEQ ID NO: 8, respectively. The cDNA fragment encodes apolypeptide of 237 amino acids. The % G+C content of the polypeptideencoding sequence is 54%. Using the SignalP software program (Nielsen etal., 1997, supra), a signal peptide of 16 residues was predicted. TheSignalP prediction is in accord with the necessity for having ahistidine reside at the N-terminus in order for proper metal binding andhence protein function to occur (See Harris et al., 2010, supra, andQuinlan et al., 2011, supra). The predicted mature protein contains 221amino acids with a predicted molecular mass of 24 kDa and a predictedisoelectric point of 6.2.

A comparative alignment of mature Family 61 amino acid sequences,without the signal peptides, was determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of EMBOSS with gap open penalty of 10, gap extensionpenalty of 0.5, and the EBLOSUM62 matrix. The alignment showed that thededuced amino acid sequence of the Trichophaea saccata Tsa2 GH61 maturepolypeptide shares 56.62% identity (excluding gaps) to the deduced aminoacid sequence of a GH61 polypeptide from Moniliophthora perniciosa FA553(SWISSPROT E2LQM6) and Coprinopsis cinerea (SWISSPROT_A8NRC9).

Example 12: Effect of Trichophaea saccata Tsa2 GH61 Polypeptide onHydrolysis of Pretreated Corn Stover

Culture broth was prepared as described in Example 10 and concentratedapproximately 20-fold as described in Example 3. PCS hydrolysisexperiments and determination of the degree of cellulose conversion wasperformed according to the procedures described in Example 3.

The result of adding increasing amounts of the Trichophaea saccata Tsa2GH61 polypeptide to the base cellulase mix are shown in FIG. 4. Additionof the Trichophaea saccata Tsa2 GH61 polypeptide provided a stimulationfactor of 1.11 at a 100% addition level.

Example 13: Cloning and Expression of a Penicillium thomii Pt1 GH61Polypeptide

Penicillium thomii was isolated from a Crocus bulb in Denmark by Prof.Jen Frisvad at the Danish Technical University (DTU) and preserved underthe accession number IBT 10776 at the IBT Culture Collection of Fungi,Danish Technical University, Denmark.

A Penicillium thomii Pt1 GH61 core fragment was cloned by PCR using thedegenerate primers shown below, which were designed to amplify aconserved core region of the GH61 polypeptide gene utilizing the Codehopprocedure (Rose et al., 1998, Nucleic Acids Res. 26: 1628-1635). GenomicDNA was isolated from Penicillium thomii according to the method inExample 1.

Primer GH61CHS3: (SEQ ID NO: 19)5'-ACCGTCGACAAGACCCAGCTCGAGTTYTTYAARAT-3' Primer GH61A_76_a:(SEQ ID NO: 20) 5'-GGCGCCGTGGAGGGCDATGATYTCRTGNC-3'

The PCR reaction (15 μl) was composed of 7.5 μl of Extensor Hi-FidelityMaster Mix (ABgene, Epsom, United Kingdom), 0.5 μl of P. thomii genomicDNA (100 ng), 0.5 μl of primer GH61CHS3 (10 mM), 0.5 μl of primerGH61A_76_a (10 mM), and 6.0 μl of deionized water. The PCR reaction wasincubated in a DNA Engine Cycler (MJ Research, Waltham, Mass., USA)programmed for:

Step 1 at 94° C. for 2 minutes;

Step 2 at 94° C. for 15 seconds;

Step 3 at 68° C. for 30 seconds; the temperature was decreased by 1° C.per cycle.

Step 4 at 68° C. for 1 minutes and 45 seconds;

Steps 2-4 were repeated for 9 cycles;

Step 6 at 94° C. for 2 minutes;

Step 7 at 94° C. for 15 seconds;

Step 8 at 58° C. for 30 seconds;

Step 9 at 68° C. for 1 minute and 45 seconds;

Step 10—repeat cycle 6 for 24 times; and

Step 11 at 68° C. for 7 minutes.

Samples were cooled to 10° C. before removal and further processing.

Five μl of the PCR reaction were analyzed by 1% agarose gelelectrophoresis using TAE buffer where an approximately 200 bp productband was observed. The band was excised from the gel, purified using anILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions, and sequenced using appropriately dilutedPCR primers for Sanger sequencing.

In order to obtain a full-length open reading frame for the GH61 corefragment obtained from the P. thomii Codehop procedure, the corefragment sequence was used to design DNA primers shown below for primerwalking using a DNA Walking SpeedUp™ Kit I (Seegene Inc., Rockville,Md., USA).

Primer PtzTCPD1: (SEQ ID NO: 21) 5'-TGATCAGCGATACCACCGAGC-3'Primer Pt-TSP1D: (SEQ ID NO: 22) 5'-CAATAGCCGTACTGTCACCGTCC-3'Primer Pt-TSP2D:  (SEQ ID NO: 23) 5'-CAGGGTGGTTTGATCAGCGATACCAC-3'

The amplification was performed according to the Kit's protocol, exceptthat the Extensor Hi Fidelity PCR Master Mix was used. The first roundof PCR with the above primers resulted in three PCR bands each of about3 kb in size. The PCR bands were sequenced with the PTSP1D primer. Theresulting sequence was assembled onto the existing previous 500 bp DNAfragment using the SeqMan sequence assembly program of the DNAStar v6.1software suite (DNA Star Inc., Madison, Wis., USA). The resulting 744 bpfragment was used to design three new DNA walking primers shown below.

Primer Pt-TSP1U: (SEQ ID NO: 24) 5'-GGACGGTGACAGTACGGCTATTG-3'Primer PtzTCP2u: (SEQ ID NO: 25) 5'-CGATGAGGTTGTCAGTTGCCCAGG-3'Primer Pt-TSP2U: (SEQ ID NO: 26) 5'-GTGGTATCGCTGATCAAACCACCCTG-3'

The PCR reaction was composed of 4 μl of P. thomii genomic DNA (50ng/μl), 1 μl of DNA Walking primer (DW-ACP 1, 2, 3, or 4), 1 μl ofprimer Pt-TSP1U (100 mM), 25 μl of 2× REDDYMIX™ (AB Gene, ABgene, Epsom,United Kingdom), which includes, buffer, dNTPs, and DNA polymerase, (ABGene, ABgene, Epsom, United Kingdom), and 18 μl of deionized water. DNAcycling conditions and the two subsequent PCR steps were performedaccording to the protocol described for the DNA Walking SpeedUp™ Kit Iwith the following primers:

2^(nd) PCR:  Primer PTZTCP2U 3^(rd) PCR:  Primer PT-TSP2U

The PCR fragments were cloned into a TA cloning vector using a pGEM®-TVector System I (Promega Corp., Madison, Wis., USA) according to themanufacturer's instructions. Ligated products were transformed into ONESHOT® TOP10 Chemically Competent E. coli cells and E. coli colonies wereselected based on blue white selection as detailed in the pGEM®-T VectorSystem I instructions. Plasmid DNA was isolated using a JETQUICK™Plasmid Purification Spin Kit according to the manufacturer'sinstructions and the pGEM plasmids were sequenced using vector primersalso detailed in the pGEM®-T Vector System I instructions. Sequenceresults were added to the SeqMan assembly described previously in thisExample and a full-length genomic sequence was obtained by the use ofthe customized primers below. The following customized primers allowedfor PCR amplification of the entire contiguous Pt1 GH61 polypeptidegenomic fragment using Penicillium thomii genomic DNA obtained accordingto Example 1. The sequence of the GH61 subgenomic fragment was 1485 bpin length.

Primer PtSeqDwn: (SEQ ID NO: 27) 5'-CCCAGCTCATCAATCGTCAGT-3'Primer PtSeqUp1: (SEQ ID NO: 28) 5'-GGTCATTGGTGATCACGACA-3'

Use of the two sequencing primers above permitted the completion of thegenomic GH61 polypeptide coding sequence (SEQ ID NO: 9). The PCRgenerated fragment containing the GH61 polypeptide coding sequence is1455 bp (including stop codon) and contains no introns.

Construction of a vector for expression of the Pt1 GH61 polypeptideencoding sequence was performed as described in Example 1, with theexception that genomic DNA from Penicillium thomii was used for PCR ofthe expression cassette. The following primers were used in theamplification:

Primer F-P33YA: (SEQ ID NO: 29)5'-GCGGAATTCACCATGTCTCTGTCTAAGATTTCTGGA-3' Primer R-P33YA:(SEQ ID NO: 30) 5'-ATATGCGGCCGCCTTCTAGTTGATGGTAATATCACGAGC-3'Bold letters represent Pt1 GH61 polypeptide coding sequence. Theunderlined sequence contains the Eco RI restriction site on the forwardPCR primer (F-P33YA) and the Not I restriction site on the reverseprimer (R-P33YA). When the primers are used in a PCR reaction with cDNAor genomic DNA from P. thomii, a fragment can be produced that can berestricted with Eco RI and Not I to produce a fragment that can becloned directionally into a suitable vector with the same restrictionsites.

Five μl of the PCR reaction were analyzed by 1% agarose gelelectrophoresis using TAE buffer where an approximately 1.4 kb productband was observed. The remaining PCR reaction was purified using anILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions. The purified PCR fragment was then digestedwith Eco RI and Not I and again purified using an ILLUSTRA™ GFX™ PCR DNAand Gel Band Purification Kit with a final elution volume of 50 μl of 10mM Tris pH 8.0.

The purified and Eco RI-Not I digested 1.4 kb PCR product was ligatedinto the Aspergillus expression vector pXYG1051 digested with Eco RI-NotI. The ligation (10.2 μl) was composed of 1 μl of Eco RI-Not I digestedpXYG1051 (10 ng/μl of TE), 8 μl of the Pt1 PCR fragment (approximately50 ng/μl), 1 μl of 10× T4 DNA ligase buffer, and 0.2 μl of T4 DNAligase. The reaction was incubated overnight at 16° C.

A 1 μl volume of the ligation reaction mixture was transformed into ONESHOT® TOP10 Chemically Competent E. coli cells (50 μl) according to themanufacturer's instructions. The transformation was plated onto LB agarplates supplemented with 100 μg of ampicillin per ml and the plates wereincubated overnight at 37° C. Six colonies were chosen that grew underselection and inoculated into 2 ml of LB medium supplemented with 100 μgof ampicillin per ml in FALCON® tubes. Plasmid DNA was isolated using aQIAprep Spin Miniprep Kit according to the manufacturer's instructions.The plasmid DNA was digested with Eco RI and Hind III and the digestsanalyzed by 1% agarose gel electrophoresis using TBE buffer, whichindicated that all six clones contained an insert of the correct size(1450 bp). The clones were then sequenced with an ABI 3730 XL GeneticAnalyzer. One error-free clone comprising the Penicillium thomii Pt1GH61 polypeptide genomic DNA sequence of SEQ ID NO: 9 was selected.

The E. coli clone was cultivated in 50 ml of LB medium supplemented with100 μg of ampicillin per ml. Plasmid DNA was isolated and purified usinga Plasmid Midi Kit according to the manufacturer's instructions. Aquantity of 1.6 μg of Pt1 GH61 plasmid DNA was used to transformAspergillus oryzae JaL355 (WO 2001/98484) according to the protocoldescribed in Example 1. Eighteen transformants were selected for furthercharacterization by SDS-PAGE analysis of the culture broths as describedin Example 1. Several transformant culture fluids contained a proteinband of 65 kDa. An explanation of the larger than predicted size of 45kDa is probably the result of glycosylation. One Aspergillus oryzaetransformant producing Pt11 GH61 polypeptide, as judged by SDS-PAGEanalysis, was chosen for further work and designated A. oryzae EXP03119.A. oryzae EXP03119 was fermented in 1000 ml Erlenmeyer shake flasks with100 ml of YP medium supplemented with 2% glucose at 26° C. for 4 dayswith agitation at 85 rpm. Several shake flasks were used to provideenough culture broth for subsequent filtration, concentration, and/orpurification of the recombinantly produced polypeptide.

An alternative method for cloning and expressing the Pt1 GH61polypeptide is described below. Based on the nucleotide sequence of SEQID NO: 9, a synthetic gene can be obtained from a number of vendors suchas Gene Art or DNA 2.0. The synthetic gene can be designed toincorporate additional DNA sequences such as restriction sites orhomologous recombination regions to facilitate cloning into anexpression vector. Using the two synthetic oligonucleotide primersF-P33YA and R-P33YA described above, a simple PCR reaction can be usedto amplify the full-length open reading frame from the synthetic gene ofSEQ ID NO: 9. The gene can then be cloned into an expression vector, forexample, as described above and expressed in a host cell, for example,Aspergillus oryzae. The GH61 polypeptide expressed in this waycorresponds to SEQ ID NO: 10.

Example 14: Characterization of the Penicillium thomii Pt1 GH61Polypeptide

The genomic DNA sequence and deduced amino acid sequence of thePenicillium thomii Pt1 GH61 polypeptide encoding sequence are shown inSEQ ID NO: 9 and SEQ ID NO: 10, respectively. The genomic DNA fragmentencodes a polypeptide of 484 amino acids. The % G+C content of thepolypeptide encoding sequence is 53.4%. Using the SignalP softwareprogram (Nielsen et al., 1997, supra), a signal peptide of 19 residueswas predicted. The SignalP prediction is in accord with the necessityfor having a histidine reside at the N-terminus in order for propermetal binding and hence protein function to occur (See Harris et al.,2010, supra, and Quinlan et al., 2011, supra). The predicted matureprotein contains 465 amino acids with a predicted molecular mass of 48kDa and a predicted isoelectric point of 4.4. The GH61 core catalyticdomain is amino acids 20 to 247 of SEQ ID NO: 10.

A comparative alignment of mature Family 61 amino acid sequences,without the signal peptides, was determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of EMBOSS with gap open penalty of 10, gap extensionpenalty of 0.5, and the EBLOSUM62 matrix. The alignment showed that thededuced amino acid sequence of the Penicillium thomii Pt1 GH61A maturepolypeptide shares 65.2% identity (excluding gaps) to the deduced aminoacid sequence of a GH61 polypeptide from Neosartorya fischeri (SWISSPROTA1 D2G7).

Example 15: Effect of Penicillium thomii Pt1 GH61 Polypeptide onHydrolysis of Pretreated Corn Stover

Culture broth was prepared as described in Example 13 and concentratedapproximately 20-fold as described in Example 3. PCS hydrolysisexperiments and determination of the degree of cellulose conversion wasperformed according to the procedures described in Example 3.

The result of adding increasing amounts of the Penicillium thomii Pt1GH61 polypeptide to the base cellulase mix are shown in FIG. 5. Additionof the Penicillium thomii Pt1 GH61 polypeptide provided a stimulationfactor of 1.11 at a 100% addition level.

The present invention is further described by the following numberedparagraphs:

[1] An isolated polypeptide having cellulolytic enhancing activity,selected from the group consisting of: (a) a polypeptide having at least60% sequence identity to the mature polypeptide of SEQ ID NO: 6 or SEQID NO: 8; at least 65% sequence identity to the mature polypeptide ofSEQ ID NO: 4; at least 70% sequence identity to the mature polypeptideof SEQ ID NO: 10; or at least 80% sequence identity to the maturepolypeptide of SEQ ID NO: 2; (b) a polypeptide encoded by apolynucleotide that hybridizes under medium-high conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, (ii) the genomic DNA sequence ofthe mature polypeptide coding sequence of SEQ ID NO: 3, SEQ ID NO: 5, orSEQ ID NO: 7, or the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) the full-length complement of (i) or(ii); (c) a polypeptide encoded by a polynucleotide having at least 60%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 5 or SEQ ID NO: 7, or the genomic DNA sequence thereof; at least 65%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 3 or the genomic DNA sequence thereof; at least 70% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 9; orat least 80% sequence identity to the mature polypeptide coding sequenceof SEQ ID NO: 1 or the cDNA sequence thereof; (d) a variant of themature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, or SEQ ID NO: 10 comprising a substitution, deletion, and/orinsertion at one or more (e.g., several) positions; and (e) a fragmentof the polypeptide of (a), (b), (c), or (d) that has cellulolyticenhancing activity.

[2] The polypeptide of paragraph 1, having at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to the mature polypeptide of SEQ ID NO: 6 or SEQ ID NO: 8; atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to the mature polypeptide of SEQ ID NO: 4; atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to the mature polypeptide of SEQ ID NO: 10; or at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to the mature polypeptide of SEQ IDNO: 2.

[3] The polypeptide of paragraph 1 or 2, which is encoded by apolynucleotide that hybridizes under medium-high, high, or very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9,(ii) the genomic DNA sequence of the mature polypeptide coding sequenceof SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, or the cDNA sequence ofthe mature polypeptide coding sequence of SEQ ID NO: 1, or (iii) thefull-length complement of (i) or (ii).

[4] The polypeptide of any of paragraphs 1-3, which is encoded by apolynucleotide having at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 5 or SEQ ID NO: 7, or thegenomic DNA sequence thereof; at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 3 or the genomic DNA sequencethereof; at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to the mature polypeptide coding sequence of SEQID NO: 9; or at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to themature polypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequencethereof.

[5] The polypeptide of any of paragraphs 1-4, comprising or consistingof SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO:10.

[6] The polypeptide of any of paragraphs 1-4, comprising or consistingof the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, or SEQ ID NO: 10.

[7] The polypeptide of paragraph 6, wherein the mature polypeptide isamino acids 21 to 322 of SEQ ID NO: 2, amino acids 18 to 234 of SEQ IDNO: 4, amino acids 24 to 233 of SEQ ID NO: 6, amino acids 17 to 237 ofSEQ ID NO: 8, or amino acids 20 to 484 of SEQ ID NO: 10.

[8] The polypeptide of paragraph 1, wherein the variant of the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,or SEQ ID NO: 10 comprises a substitution, deletion, and/or insertion atone or more (e.g., several) positions.

[9] The polypeptide of any of paragraphs 1-4, which is a fragment of SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10,wherein the fragment has cellulolytic enhancing activity.

[10] A composition comprising the polypeptide of any of paragraphs 1-9.

[11] An isolated polynucleotide encoding the polypeptide of any ofparagraphs 1-9.

[12] A nucleic acid construct or expression vector comprising thepolynucleotide of paragraph 11 operably linked to one or more controlsequences that direct the production of the polypeptide in an expressionhost.

[13] A recombinant host cell comprising the polynucleotide of paragraph11 operably linked to one or more control sequences that direct theproduction of the polypeptide.

[14] A method of producing the polypeptide of any of paragraphs 1-9,comprising cultivating a cell, which in its wild-type form produces thepolypeptide, under conditions conducive for production of thepolypeptide.

[15] The method of paragraph 14, further comprising recovering thepolypeptide.

[16] A method of producing a polypeptide having cellulolytic enhancingactivity, comprising cultivating the host cell of paragraph 13 underconditions conducive for production of the polypeptide.

[17] The method of paragraph 16, further comprising recovering thepolypeptide.

[18] A transgenic plant, plant part or plant cell transformed with apolynucleotide encoding the polypeptide of any of paragraphs 1-9.

[19] A method of producing a polypeptide having cellulolytic enhancingactivity, comprising cultivating the transgenic plant or plant cell ofparagraph 18 under conditions conducive for production of thepolypeptide.

[20] The method of paragraph 19, further comprising recovering thepolypeptide.

[21] A method of producing a mutant of a parent cell, comprisinginactivating a polynucleotide encoding the polypeptide of any ofparagraphs 1-9, which results in the mutant producing less of thepolypeptide than the parent cell.

[22] A mutant cell produced by the method of paragraph 21.

[23] The mutant cell of paragraph 22, further comprising a gene encodinga native or heterologous protein.

[24] A method of producing a protein, comprising cultivating the mutantcell of paragraph 22 or 23 under conditions conducive for production ofthe protein.

[25] The method of paragraph 24, further comprising recovering theprotein.

[26] A double-stranded inhibitory RNA (dsRNA) molecule comprising asubsequence of the polynucleotide of paragraph 11, wherein optionallythe dsRNA is a siRNA or a miRNA molecule.

[27] The double-stranded inhibitory RNA (dsRNA) molecule of paragraph26, which is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or moreduplex nucleotides in length.

[28] A method of inhibiting the expression of a polypeptide havingcellulolytic enhancing activity in a cell, comprising administering tothe cell or expressing in the cell the double-stranded inhibitory RNA(dsRNA) molecule, wherein the dsRNA of paragraph 26 or 27.

[29] A cell produced by the method of paragraph 28.

[30] The cell of paragraph 29, further comprising a gene encoding anative or heterologous protein.

[31] A method of producing a protein, comprising cultivating the cell ofparagraph 29 or 30 under conditions conducive for production of theprotein.

[32] The method of paragraph 31, further comprising recovering theprotein.

[33] An isolated polynucleotide encoding a signal peptide comprising orconsisting of amino acids 1 to 20 of SEQ ID NO: 2, amino acids 1 to 17of SEQ ID NO: 4, amino acids 1 to 23 of SEQ ID NO: 6, amino acids 1 to16 of SEQ ID NO: 8, or amino acids 1 to 19 of SEQ ID NO: 10.

[34] A nucleic acid construct or expression vector comprising a geneencoding a protein operably linked to the polynucleotide of paragraph33, wherein the gene is foreign to the polynucleotide encoding thesignal peptide.

[35] A recombinant host cell comprising a gene encoding a proteinoperably linked to the polynucleotide of paragraph 33, wherein the geneis foreign to the polynucleotide encoding the signal peptide.

[36] A method of producing a protein, comprising cultivating arecombinant host cell comprising a gene encoding a protein operablylinked to the polynucleotide of paragraph 33, wherein the gene isforeign to the polynucleotide encoding the signal peptide, underconditions conducive for production of the protein.

[37] The method of paragraph 36, further comprising recovering theprotein.

[38] A method for degrading or converting a cellulosic material,comprising: treating the cellulosic material with an enzyme compositionin the presence of the polypeptide having cellulolytic enhancingactivity of any of paragraphs 1-9.

[39] The method of paragraph 38, wherein the cellulosic material ispretreated.

[40] The method of paragraph 38 or 39, wherein the enzyme compositioncomprises one or more (several) enzymes selected from the groupconsisting of a cellulase, a GH61 polypeptide having cellulolyticenhancing activity, a hemicellulase, an expansin, an esterase, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin.

[41] The method of paragraph 40, wherein the cellulase is one or more(several) enzymes selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[42] The method of paragraph 40, wherein the hemicellulase is one ormore (several) enzymes selected from the group consisting of a xylanase,an acetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[43] The method of any of paragraphs 38-42, further comprisingrecovering the degraded cellulosic material.

[44] The method of paragraph 43, wherein the degraded cellulosicmaterial is a sugar.

[45] The method of paragraph 44, wherein the sugar is selected from thegroup consisting of glucose, xylose, mannose, galactose, and arabinose.

[46] A method for producing a fermentation product, comprising: (a)saccharifying a cellulosic material with an enzyme composition in thepresence of the polypeptide having cellulolytic enhancing activity ofany of paragraphs 1-9; (b) fermenting the saccharified cellulosicmaterial with one or more fermenting microorganisms to produce thefermentation product; and (c) recovering the fermentation product fromthe fermentation.

[47] The method of paragraph 46, wherein the cellulosic material ispretreated.

[48] The method of paragraph 46 or 47, wherein the enzyme compositioncomprises one or more (several) enzymes selected from the groupconsisting of a cellulase, a GH61 polypeptide having cellulolyticenhancing activity, a hemicellulase, an expansin, an esterase, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin.

[49] The method of paragraph 48, wherein the cellulase is one or more(several) enzymes selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[50] The method of paragraph 48, wherein the hemicellulase is one ormore (several) enzymes selected from the group consisting of a xylanase,an acetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[51] The method of any of paragraphs 46-50, wherein steps (a) and (b)are performed simultaneously in a simultaneous saccharification andfermentation.

[52] The method of any of paragraphs 46-51, wherein the fermentationproduct is an alcohol, an organic acid, a ketone, an amino acid, analkane, a cycloalkane, an alkene, isoprene, polyketide, or a gas.

[53] A method of fermenting a cellulosic material, comprising:fermenting the cellulosic material with one or more fermentingmicroorganisms, wherein the cellulosic material is saccharified with anenzyme composition in the presence of the polypeptide havingcellulolytic enhancing activity of any of paragraphs 1-9.

[54] The method of paragraph 53, wherein the fermenting of thecellulosic material produces a fermentation product.

[55] The method of paragraph 54, further comprising recovering thefermentation product from the fermentation.

[56] The method of any of paragraphs 53-55, wherein the cellulosicmaterial is pretreated before saccharification.

[57] The method of any of paragraphs 53-56, wherein the enzymecomposition comprises one or more enzymes selected from the groupconsisting of a cellulase, a GH61 polypeptide having cellulolyticenhancing activity, a hemicellulase, an expansin, an esterase, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin.

[58] The method of paragraph 57, wherein the cellulase is one or more(several) enzymes selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[59] The method of paragraph 57, wherein the hemicellulase is one ormore (several) enzymes selected from the group consisting of a xylanase,an acetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[60] The method of any of paragraphs 54-59, wherein the fermentationproduct is an alcohol, an organic acid, a ketone, an amino acid, analkane, a cycloalkane, an alkene, isoprene, polyketide, or a gas.

[61] A whole broth formulation or cell culture composition comprisingthe polypeptide of any of claims 1-9.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1-20. (canceled)
 21. A method for degrading a cellulosic material,comprising: treating the cellulosic material with an enzyme compositioncomprising a GH61 polypeptide having cellulolytic enhancing activity,wherein the GH61 polypeptide having cellulolytic enhancing activity isselected from the group consisting of: (a) a GH61 polypeptide having atleast 90% sequence identity to amino acids 21 to 322 of the polypeptideof SEQ ID NO: 2, amino acids 18 to 234 of the polypeptide of SEQ ID NO:4, amino acids 24 to 233 of the polypeptide of SEQ ID NO: 6, amino acids17 to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484of the polypeptide of SEQ ID NO: 10; (b) a GH61 polypeptide encoded by apolynucleotide that hybridizes under high stringency conditions with thefull-length complement of nucleotides 61 to 969 of the polynucleotide ofSEQ ID NO: 1, nucleotides 52 to 702 of the polynucleotide of SEQ ID NO:3, nucleotides 70 to 699 of the polynucleotide of SEQ ID NO: 5,nucleotides 49 to 711 of the polynucleotide of SEQ ID NO: 7, ornucleotides 58 to 1452 of the polynucleotide of SEQ ID NO: 9, whereinhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, and washing threetimes each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.; (c) a GH61polypeptide encoded by (i) a polynucleotide having at least 90% sequenceidentity to nucleotides 61 to 969 of the polynucleotide of SEQ ID NO: 1,nucleotides 52 to 702 of the polynucleotide of SEQ ID NO: 3, nucleotides70 to 699 of the polynucleotide of SEQ ID NO: 5, nucleotides 49 to 711of the polynucleotide of SEQ ID NO: 7, or nucleotides 58 to 1452 of thepolynucleotide of SEQ ID NO: 9; and (d) a fragment of the GH61polypeptide of (a), (b), or (c), wherein the fragment has cellulolyticenhancing activity.
 22. The method of claim 21, wherein the cellulosicmaterial is pretreated.
 23. The method of claim 21, wherein the enzymecomposition further comprises one or more enzymes selected from thegroup consisting of a cellulase, a GH61 polypeptide having cellulolyticenhancing activity, a hemicellulase, an expansin, an esterase, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin.
 24. The method of claim 21, further comprisingrecovering the degraded cellulosic material.
 25. The method of claim 24,wherein the degraded cellulosic material is a sugar selected from thegroup consisting of glucose, xylose, mannose, galactose, and arabinose.26. The method of claim 21, wherein the GH61 polypeptide havingcellulolytic enhancing activity has at least 95% sequence identity toamino acids 21 to 322 of the polypeptide of SEQ ID NO: 2, amino acids 18to 234 of the polypeptide of SEQ ID NO: 4, amino acids 24 to 233 of thepolypeptide of SEQ ID NO: 6, amino acids 17 to 237 of the polypeptide ofSEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO:10.
 27. The method of claim 21, wherein the GH61 polypeptide havingcellulolytic enhancing activity comprises (i) the polypeptide of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10; (ii)amino acids 21 to 322 of the polypeptide of SEQ ID NO: 2, amino acids 18to 234 of the polypeptide of SEQ ID NO: 4, amino acids 24 to 233 of thepolypeptide of SEQ ID NO: 6, amino acids 17 to 237 of the polypeptide ofSEQ ID NO: 8, or amino acids 20 to 484 of the polypeptide of SEQ ID NO:10; or (iii) a fragment of (i) or (ii) having cellulolytic enhancingactivity.
 28. The method of claim 21, wherein the GH61 polypeptidehaving cellulolytic enhancing activity comprises amino acids 18 to 234of the polypeptide of SEQ ID NO: 4, amino acids 17 to 237 of thepolypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of the polypeptideof SEQ ID NO:
 10. 29. A method for producing a fermentation product,comprising: (a) saccharifying a cellulosic material with an enzymecomposition comprising a GH61 polypeptide having cellulolytic enhancingactivity, wherein the the GH61 polypeptide having cellulolytic enhancingactivity is selected from the group consisting of: (i) a GH61polypeptide having at least 90% sequence identity to amino acids 21 to322 of the polypeptide of SEQ ID NO: 2, amino acids 18 to 234 of thepolypeptide of SEQ ID NO: 4, amino acids 24 to 233 of the polypeptide ofSEQ ID NO: 6, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8,or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10; (ii) aGH61 polypeptide encoded by a polynucleotide that hybridizes under highstringency conditions with the full-length complement of nucleotides 61to 969 of the polynucleotide of SEQ ID NO: 1, nucleotides 52 to 702 ofthe polynucleotide of SEQ ID NO: 3, nucleotides 70 to 699 of thepolynucleotide of SEQ ID NO: 5, nucleotides 49 to 711 of thepolynucleotide of SEQ ID NO: 7, or nucleotides 58 to 1452 of thepolynucleotide of SEQ ID NO: 9, wherein high stringency conditions aredefined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3%SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50%formamide, and washing three times each for 15 minutes using 2×SSC, 0.2%SDS at 65° C.; (iii) a GH61 polypeptide encoded by (i) a polynucleotidehaving at least 90% sequence identity to nucleotides 61 to 969 of thepolynucleotide of SEQ ID NO: 1, nucleotides 52 to 702 of thepolynucleotide of SEQ ID NO: 3, nucleotides 70 to 699 of thepolynucleotide of SEQ ID NO: 5, nucleotides 49 to 711 of thepolynucleotide of SEQ ID NO: 7, or nucleotides 58 to 1452 of thepolynucleotide of SEQ ID NO: 9; and (iv) a fragment of the GH61polypeptide of (i), (ii), or (iii), wherein the fragment hascellulolytic enhancing activity; (b) fermenting the saccharifiedcellulosic material with one or more fermenting microorganisms toproduce the fermentation product; and (c) recovering the fermentationproduct from the fermentation.
 30. The method of claim 29, wherein thecellulosic material is pretreated.
 31. The method of claim 29, whereinthe enzyme composition further comprises one or more enzymes selectedfrom the group consisting of a cellulase, a GH61 polypeptide havingcellulolytic enhancing activity, a hemicellulase, an expansin, anesterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.
 32. The method of claim 29, wherein steps (a)and (b) are performed simultaneously in a simultaneous saccharificationand fermentation.
 33. The method of claim 29, wherein the fermentationproduct is an alcohol, an organic acid, a ketone, an amino acid, analkane, a cycloalkane, an alkene, isoprene, polyketide, or a gas. 34.The method of claim 29, wherein the GH61 polypeptide having cellulolyticenhancing activity has at least 95% sequence identity to amino acids 21to 322 of the polypeptide of SEQ ID NO: 2, amino acids 18 to 234 of thepolypeptide of SEQ ID NO: 4, amino acids 24 to 233 of the polypeptide ofSEQ ID NO: 6, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8,or amino acids 20 to 484 of the polypeptide of SEQ ID NO:
 10. 35. Themethod of claim 29, wherein the GH61 polypeptide having cellulolyticenhancing activity comprises (i) the polypeptide of SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10; (ii) amino acids 21to 322 of the polypeptide of SEQ ID NO: 2, amino acids 18 to 234 of thepolypeptide of SEQ ID NO: 4, amino acids 24 to 233 of the polypeptide ofSEQ ID NO: 6, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8,or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10; or (iii) afragment of (i) or (ii) having cellulolytic enhancing activity.
 36. Themethod of claim 29, wherein the GH61 polypeptide having cellulolyticenhancing activity comprises amino acids 21 to 322 of the polypeptide ofSEQ ID NO: 2, amino acids 18 to 234 of the polypeptide of SEQ ID NO: 4,amino acids 24 to 233 of the polypeptide of SEQ ID NO: 6, amino acids 17to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 ofthe polypeptide of SEQ ID NO:
 10. 37. A method of fermenting acellulosic material, comprising: fermenting the cellulosic material withone or more fermenting microorganisms, wherein the cellulosic materialis saccharified with an enzyme composition comprising a GH61 polypeptidehaving cellulolytic enhancing activity, wherein the fermenting of thecellulosic material produces a fermentation product, and wherein theGH61 polypeptide having cellulolytic enhancing activity is selected fromthe group consisting of: (a) a GH61 polypeptide having at least 90%sequence identity to amino acids 21 to 322 of the polypeptide of SEQ IDNO: 2, amino acids 18 to 234 of the polypeptide of SEQ ID NO: 4, aminoacids 24 to 233 of the polypeptide of SEQ ID NO: 6, amino acids 17 to237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 of thepolypeptide of SEQ ID NO: 10; (b) a GH61 polypeptide encoded by apolynucleotide that hybridizes under high stringency conditions with thefull-length complement of nucleotides 61 to 969 of the polynucleotide ofSEQ ID NO: 1, nucleotides 52 to 702 of the polynucleotide of SEQ ID NO:3, nucleotides 70 to 699 of the polynucleotide of SEQ ID NO: 5,nucleotides 49 to 711 of the polynucleotide of SEQ ID NO: 7, ornucleotides 58 to 1452 of the polynucleotide of SEQ ID NO: 9, whereinhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, and washing threetimes each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.; (c) a GH61polypeptide encoded by (i) a polynucleotide having at least 90% sequenceidentity to nucleotides 61 to 969 of the polynucleotide of SEQ ID NO: 1,nucleotides 52 to 702 of the polynucleotide of SEQ ID NO: 3, nucleotides70 to 699 of the polynucleotide of SEQ ID NO: 5, nucleotides 49 to 711of the polynucleotide of SEQ ID NO: 7, or nucleotides 58 to 1452 of thepolynucleotide of SEQ ID NO: 9; and (d) a fragment of the GH61polypeptide of (a), (b), or (c), wherein the fragment has cellulolyticenhancing activity.
 38. The method of claim 37, wherein the cellulosicmaterial is pretreated before saccharification.
 39. The method of claim37, wherein the enzyme composition further comprises one or more enzymesselected from the group consisting of a cellulase, a GH61 polypeptidehaving cellulolytic enhancing activity, a hemicellulase, an expansin, anesterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.
 40. The method of claim 37, furthercomprising recovering the fermentation product from the fermentation.41. The method of claim 40, wherein the fermentation product is analcohol, an organic acid, a ketone, an amino acid, an alkane, acycloalkane, an alkene, isoprene, polyketide, or a gas.
 42. The methodof claim 37, wherein the GH61 polypeptide having cellulolytic enhancingactivity has at least 95% sequence identity to amino acids 21 to 322 ofthe polypeptide of SEQ ID NO: 2, amino acids 18 to 234 of thepolypeptide of SEQ ID NO: 4, amino acids 24 to 233 of the polypeptide ofSEQ ID NO: 6, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8,or amino acids 20 to 484 of the polypeptide of SEQ ID NO:
 10. 43. Themethod of claim 37, wherein the GH61 polypeptide having cellulolyticenhancing activity comprises (i) the polypeptide of SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO; 6, SEQ ID NO: 8, or SEQ ID NO: 10; (ii) amino acids 21to 322 of the polypeptide of SEQ ID NO: 2, amino acids 18 to 234 of thepolypeptide of SEQ ID NO: 4, amino acids 24 to 233 of the polypeptide ofSEQ ID NO: 6, amino acids 17 to 237 of the polypeptide of SEQ ID NO: 8,or amino acids 20 to 484 of the polypeptide of SEQ ID NO: 10; or (iii) afragment of (i) or (ii) having cellulolytic enhancing activity.
 44. Themethod of claim 37, wherein the GH61 polypeptide having cellulolyticenhancing activity comprises amino acids 21 to 322 of the polypeptide ofSEQ ID NO: 2, amino acids 18 to 234 of the polypeptide of SEQ ID NO: 4,amino acids 24 to 233 of the polypeptide of SEQ ID NO: 6, amino acids 17to 237 of the polypeptide of SEQ ID NO: 8, or amino acids 20 to 484 ofthe polypeptide of SEQ ID NO: 10.